EP3452589A1 - Method and system for magnetic extraction of components in a liquid sample - Google Patents

Method and system for magnetic extraction of components in a liquid sample

Info

Publication number
EP3452589A1
EP3452589A1 EP16801505.5A EP16801505A EP3452589A1 EP 3452589 A1 EP3452589 A1 EP 3452589A1 EP 16801505 A EP16801505 A EP 16801505A EP 3452589 A1 EP3452589 A1 EP 3452589A1
Authority
EP
European Patent Office
Prior art keywords
pipette
cone
phase
holder
particles
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP16801505.5A
Other languages
German (de)
French (fr)
Other versions
EP3452589B1 (en
Inventor
David ALIX
Edgar MINASSIAN
Jean-Claude Raymond
Philippe Wandels
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biomerieux SA
Original Assignee
Biomerieux SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from EP16168001.2A external-priority patent/EP3241901B1/en
Application filed by Biomerieux SA filed Critical Biomerieux SA
Publication of EP3452589A1 publication Critical patent/EP3452589A1/en
Application granted granted Critical
Publication of EP3452589B1 publication Critical patent/EP3452589B1/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/021—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
    • B01L3/0217—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids of the plunger pump type
    • B01L3/0231—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids of the plunger pump type having several coaxial pistons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0275—Interchangeable or disposable dispensing tips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50853—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates with covers or lids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
    • B01L9/523—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips for multisample carriers, e.g. used for microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/54—Supports specially adapted for pipettes and burettes
    • B01L9/543—Supports specially adapted for pipettes and burettes for disposable pipette tips, e.g. racks or cassettes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0609—Holders integrated in container to position an object
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids

Definitions

  • the invention relates to the field of extracting components contained in a solution using magnetic particles.
  • the invention finds particular application in the field of biological sample preparation, especially in the implementation of in vitro diagnostics, by the capture of analytes of biological origin (nucleic acids, microorganisms, proteins, peptides, etc.). ) present in a solution.
  • analytes of biological origin nucleic acids, microorganisms, proteins, peptides, etc.
  • the "BOOM®” technology consists in introducing into a liquid sample magnetic particles capable of binding with components of interest, then to separate the magnetic particles from the sample using one or more magnets. The particles thus captured can then undergo further processing, for example to release their components in a recovery solution. Because of the efficiency of this technique, many devices have been developed and marketed, especially for DNA and RNA, 7-9, both manual devices (for example the applicant's NucliSENS-miniMAG®) and devices automated systems (NucliSENS-easyMAG® of the applicant). These automated devices, however, suffer from various limitations.
  • a first limitation concerns their versatility and their size. Indeed, these devices are most often heavy and bulky automata that are designed to implement a non-modifiable treatment sequence by the user. An automaton is thus designed for a single type of extraction, for example designed for the purification of nucleic acids but unable to implement a magnetic immuno-concentration.
  • a second limitation relates to the injection and suction circuits of the different liquids used during the extraction.
  • the number of liquids being important, this involves circuits that are also numerous and / or complex.
  • these injection / suction circuits must be regularly cleaned, which involves the disabling of the devices.
  • a third limitation relates to the brewing operations that are implemented to obtain the homogeneity of the sample comprising the magnetic particles before capture, to maximize the capture by them of the analytes of interest or to effectively wash the magnetic particles. This type of mixing usually requires complex mechanisms, for example based on mobile magnets that set the magnetic particles in motion.
  • the fourth limitation concerns the different liquids used during extraction.
  • the steps implemented for the extraction are carried out in one or from a single container.
  • this container sets an identical volume for all the liquids involved (e.g. the sample, the different washing solutions, the elution solution, etc.), which limits the overall efficiency of the extraction process. Indeed, some treatments (e.g. washing) require large volumes to be fully effective while other treatments are content with a small volume of liquid (e.g., elution).
  • the object of the present invention is to provide a method of extracting components in a liquid sample using magnetic particles which offers a great freedom in the choice of liquid volumes, in particular up to 10 ml, used during extraction.
  • the subject of the invention is a process for extracting components contained in a biological sample in liquid form, said components being capable of being fixed on magnetic particles, the process comprising:
  • a suction phase of the mixture from a well in a tubular pipette cone comprising a tip intended for liquid pipetting
  • a phase of capture of the magnetic particles on an inner wall of the pipette cone by applying a first magnetic field to the pipette cone, said field being able to attract and hold the magnetic particles in a predetermined zone of the pipette cone, so-called of "capture", above the tip of it;
  • the invention takes advantage of a pipette cone in which suction and discharge cycles can be performed by dipping its tip in a well.
  • suction and discharge cycles can be performed by dipping its tip in a well.
  • the volume of the recovery solution may therefore be small if necessary.
  • the volume of the recovery solution is thus independent of the volume of the sample and the volume of the pipette cone in which the extraction is carried out. Thanks to the invention, it is therefore possible to optimize each volume of liquid used, and thus optimize extraction.
  • the displacement of the first magnetic field consists in moving the pipette cone parallel to a longitudinal axis of said cone, and in keeping the first magnetic field constant, the longitudinal axis of the pipette cone remaining equidistant from the first one; magnetic field when moving the pipette cone.
  • the migration of the particles can be carried out in a simple manner by moving the pipette cone relative to, for example, a permanent magnet.
  • the transfer phase comprises:
  • the second magnetic field is produced by a magnet positioned partially or wholly under the tip of the pipette cone.
  • the first magnetic field applied to the pipette cone is disabled upon application of the second magnetic field.
  • the second magnetic field makes it possible to simply attract the particles into the recovery well, which increases the rate of recovery of the magnetic particles in the recovery well, as well as the number of particles recovered.
  • the second magnetic field automatically captures the magnetic particles in the recovery well. For example, if the recovery solution is an eluent, the particle-bound components have been released and a technician can directly pipet the solution which is devoid of magnetic particles.
  • the transfer phase comprises the deactivation of the first magnetic field followed by the application of suction and discharge cycles of the solution of the recovery well in the tip of the pipette cone, said application comprising:
  • the first phase effectively breaks up the cluster of particles captured on the pipette cone, also called "pellet", and thus resuspend the particles in the recovery solution.
  • the second phase allows to continue stirring the solution while not preventing the migration of particles under the effect of the magnetic field. This makes it possible to further increase the recovery rate and the number of particles recovered in the recovery well.
  • the solution is an eluent whose function is to release the components captured by the magnetic particles, these cycles have the effect of stirring the particles in the eluent, which increases the effectiveness of the eluent, in particular when using an elution solution in the stall of magnetic particle analytes without heating step.
  • the method comprises, prior to the capture phase, a stirring phase of the mixture contained in the pipette cone by the application of at least one suction and discharge cycle of said mixture in the cone pipette. Due to the cone geometry, tubular shape, it is possible to obtain a large volume flow rate to the section of the cone, and therefore effective mixing. In addition, there is great turbulence in the cone naturally generated by the flow of liquid, turbulence that increase the efficiency of the brewing. Advantageously, a disposable accessory is provided in the cone to increase this effect.
  • the method comprises, prior to the transfer phase, at least one washing phase of the particles captured on the inner wall of the pipette cone by: - deactivating the magnetic field;
  • the release of the captured particles comprises a phase of applying the cycles so as to reciprocate a meniscus of said solution on a pellet of particles captured in the pipette cone. and-coming of said meniscus being made on a portion of the cone lower than the total length of the pipette cone.
  • the release of the captured particles comprises a second phase of application of the cycles so as to completely suck up and discharge the washing solution of the cone.
  • the frequency of application of the cycles of the second phase is lower than the frequency of application of the cycles of the first phase.
  • the method comprises at least two washing phases implemented in two separate washing solutions.
  • the components contained in the biological sample are nucleic acids (eg DNA, RNA).
  • the components contained in the biological sample are microorganisms (eg bacteria, fungi, yeasts), and wherein the method comprises a single capture phase and a single wash phase.
  • the mixture of the sample with the magnetic particles has a volume greater than 1 milliliter, and preferably greater than or equal to 2 milliliters, and in which the volume of the recovery well is less than or equal to 200 micro liters, and preferably less than or equal to 100 micro liters.
  • the method comprises, prior to the transfer phase, at least one washing phase of the particles captured on the inner wall of the pipette cone by: drawing the washing solution into said pipette cone;
  • the modulation of the magnetic field induces a reorganization of the particle pellet captured on the wall of the cone.
  • the base can change shape, spread, slide or "roll" on the wall of the pipette cone. This reorganization of the base makes it possible to further increase the effectiveness of the washing, and all the more so since this reorganization can be implemented in conjunction with suction and discharge cycles of the washing solution.
  • the modulation of the first magnetic field is carried out:
  • the modulation is obtained simply, for example by a technician who slides a bar of magnets or by a PLC which puts a simple mechanism of translation of a bar of magnets.
  • the volume of the pipette cone is at least ten times greater than the volume of the recovery well.
  • the volume of the mixture is at least three times greater than the volume of the pipette cone.
  • the components belong to the group consisting of single-stranded or double-stranded nucleic acids (DNA and / or RNA), microorganisms, proteins, and peptides. The components consist of all other types of molecules depending on the functionalization given to the magnetic particles.
  • the object of the present invention is also to provide a device for implementing the method just described, which is compact and easy to use by a laboratory technician.
  • the invention also relates to a pipette holder comprising:
  • a pipette holder comprising a first housing in which is insertable, advantageously removably, a pipette equipped with at least one tubular pipette cone comprising a tip for liquid pipetting, the first housing being open on the recess of the base, the pipette holder being movable in translation relative to the base in a direction parallel to an axis of the pipette cones and movable between a first position in which the tip of each pipette cone is housed in a well of the support of well and at least a second position in which said tip is outside said well;
  • a second housing adapted to releasably receive a magnetized part, the second housing facing each of the pipette tips in a position above the tip thereof when the pipette holder is in the first position, and the second housing faces the tip of the pipette cone when the pipette holder is in the second position.
  • the pipette holder receives a pipette and the technician implements the steps of the extraction process, up / down the pipette, in particular to migrate the particle pellet in the tip of the cone (s). pipette, introducing wells (in the form of plate, bar, etc.) into the base, and actuating the pipette.
  • the pipette holder which is compact and transportable, also allows semi-automation of the extraction process when an electronic pipette is used.
  • a pipette comprises indeed suction and discharge circuits in each pipette cone equipping it, and a microprocessor-based electronic circuit.
  • This electronic circuit controls the suction / discharge circuits according to instructions entered by the technician by means of an interface fitted to the pipette and / or a computer / tablet / smartphone connected to the pipette (eg via a wireless link Of type bluetooth), etc.
  • These instructions consist, for example, of instructions for suction / discharge cycles and / or of a choice of a particular protocol pre-recorded in the pipette.
  • the electronic pipette is programmable, great versatility is furthermore obtained in the definition of the extraction process, which can be adapted according to a particular desired magnetic capture (eg: purification of nucleic acids, magnetic immunoconcentration ,. ..).
  • a suitable protocol for the targeted extraction can be recorded in the pipette, the protocol being defined in terms of the number of suction and discharge cycles, the sequence of cycles, the frequency of cycles, the duration between cycles. , defined volumes, etc.
  • the first housing includes an opening for the front insertion and withdrawal of the pipette into the first housing of the pipette holder.
  • the front insertion and removal of the pipette and cones in place minimizes the risk of touching the pipette holder with cone tips, and thus the risk of contamination of the pipette holder.
  • the second housing is made in the base.
  • the pipette holder has a third housing in which the magnetized piece is removable to face each pipette cone at a position above the tip of said cone when the pipette holder is in the second position.
  • the magnetized part e.g. comprising one or more permanent magnets
  • the magnetized part is integral with the pipette tips and therefore follows their translational movement relative to the base. During such movements, the magnetized part thus retains the pellets of magnetic particles fixed in the cones.
  • the technician can for example mount the pipette to more easily move a well support in the base without the risk of moving the pellets of particles in the cones.
  • the second and third housings communicate, and the pipette support comprises means able to removably hold the magnetized part in the third housing.
  • the technician can detach the magnetized piece from pipette holder which then automatically takes place in the base by falling into the second housing. This detachment in particular takes place for the migration operation of the magnetic particles in the tips of the cones.
  • the base comprises at least one rotating gear wheel
  • the pipette holder comprises a rack cooperating with the gearwheel to translate the pipette holder relative to the base during rotation of the gearwheel.
  • the pipette holder comprises a device for locking and unlocking the pipette holder in the first position.
  • the pipette holder comprises at least one handle secured to the toothed wheel to rotate it and adapted to be removably attached to a handle secured to the gear wheel of another pipette holder, which therefore allows to increase the number of pipette tips during the extraction process.
  • the invention also relates to a system for extracting components contained in a biological sample in liquid form, said components being capable of being fixed on magnetic particles, the system comprising:
  • a pipette equipped with at least one tubular pipette cone comprising a tip intended for liquid pipetting and a suction and discharge circuit in each pipette cone;
  • a pipette holder comprising:
  • a pipette support comprising a first housing in which the pipette is inserted, advantageously removably, the first housing being open on the recess of the base, the pipette support being movable in translation relative to the base in a direction parallel to an axis of the pipette cones and movable between a first position in which the tip of each pipette cone is housed in a well of the well holder and at least a second position in which said tip is out of said well;
  • the pipette holder is in accordance with the pipette holder described above.
  • the invention also aims to provide a well support for the migration of magnetic particles from tips of pipette cones in recovery wells.
  • the invention also relates to a well support, comprising a part in which are formed recesses for receiving the wells, and at least one magnet facing each of the recesses formed in said part.
  • Figure 1 is a perspective view of an extraction system according to the invention
  • FIGS. 2A and 2B are front and perspective views of an electronic pipette and its removable pipette tips
  • FIGS. 3A and 3B are perspective and front views of a pipette holder according to the invention.
  • FIGS. 4A and 4B are a detail sectional view of the pipette holder of FIG. 3, respectively along the planes A-A and B-B of FIG. 3B;
  • Figure 5 is a perspective view of a deepwell plate including wells
  • Figures 6A and 6B are perspective views of a magnetic rack and PCR elution tubes that can be accommodated in the rack;
  • Figure 7 is a front view of a magnetized part according to the invention.
  • Figure 8 is a flow chart of an extraction process according to the invention.
  • Figure 9 is a photograph of the pipette tips of a system according to the invention with pellets of magnetic particles placed about halfway up the cones;
  • FIG. 11 is a photograph of PCR elution tubes in which the magnetic particles have been recovered:
  • Figures 12 and 13 illustrate a second embodiment of the pipette holder according to the invention
  • Figure 14 is a perspective view of two systems according to Figure 1 coupled by means of rotation handles;
  • Figure 15 is a schematic view illustrating the reciprocation of a meniscus of a solution on a particle pellet to detach the latter from the wall of a pipette cone. Except for Figure 15, the description is made in relation to plans and photographs on a reduced scale of a real system.
  • a system 10 for extracting (FIG. 1) components contained in a liquid sample comprises an electronic pipette 12 (FIG. 2), a pipette holder 14 (FIGS. ) in which the pipette 12 is housed, one or more well supports 18a, 18b (FIGS. 5 and 6) can each be housed in the pipette holder 14, and a first magnetized piece 16 (FIG. 7).
  • the pipette 12 which is portable, comprises a row of pipette tips 20, and a body 22 on which the cones 20 are mounted (FIG. 2A).
  • a liquid suction / discharge circuit in the cones 20 for example a set of pistons actuated by an electric motor
  • an electronic control circuit of the suction / discharge circuit for example a set of pistons actuated by an electric motor
  • the electronic circuit which comprises for example a microprocessor and one or more computer memories, is programmable, and embeds instructions for the implementation of one or more pipetting protocols, each protocol comprising one or more steps.
  • the electronic circuit also comprises a man-machine interface 24 housed in a handle 26 of the body 22, the interface comprising a set of selection and navigation buttons 28 and a display screen 30 allowing the display and selection of different control protocols. pipetting recorded.
  • the user can in particular program the electronic pipette 12, eg by downloading instructions therefrom from a computer connected to the pipette 12 through a wireless link, for example Bluetooth.
  • the user can also select, via the interface 24, a prerecorded protocol.
  • the pipette 12 is also able to suck up a predefined volume of liquid in each of the cones 20, to discharge a predefined volume from each of the cones, to implement automatic suction / discharge cycles of variable duration and frequency. As illustrated more particularly in FIG.
  • each cone 20 has at its open pipetting end a tapered profile 21, or "tip". This portion 21 has a reduced section in a plane perpendicular to the X axis, which allows its introduction facilitated in containers or wells as is known per se.
  • the electronic pipette 12 is for example the "VIAFLO II 8-channel" model marketed by the company ⁇ INTEGRA Biosciences AG, Switzerland, the model of which elements are described in the patent applications US 2009/071266, US 2009/074622, US2011076205 and US 2008/095671.
  • the pipette holder 14 comprises meanwhile (FIGS. 3A and 3B) a base 34 intended to be placed on a work surface (eg a laboratory table or bench) and a mobile part 36 with respect to the base. 34.
  • the mobile part 36 also called “pipette holder”, comprises a housing 38 in which the pipette 12 can be removably inserted and kept stationary as illustrated in FIG. 1.
  • the support 36 comprises one or more racks 40, for example two in number, meshing with gear wheels 42 mounted on a shaft 44 rotatable and housed in the base 34.
  • One or more rods 46 are furthermore fixed to the support 36 (or in the base 34) and slide in orifices of the base 34 (or in the support 36), in order to guide the support 36 in its translational movement.
  • One or two rotational handles 50 are further attached to the end of the shaft 44 to allow the user to easily turn the latter according to the arrows 52 ( Figure 1) and thus raise and lower the pipette holder 36 as as illustrated by the arrows 54 ( Figure 1).
  • the translational movement of the pipette holder 36 relative to the base 34 is therefore parallel to the axis X of the cones 20 when the pipette 12 is placed in the housing 38 of the support 36, and therefore parallel to the direction of gravity when the base 34 is placed on a horizontal work plane, so that the support 36 "up” or "down".
  • the base 34 is open on its front face 56 to allow the introduction and removal of the well supports 18a, 18b, thus defining a housing for the latter.
  • This housing is open on its upper part to allow the cones 20 of the pipette 12 to reach said well supports when the pipette goes down.
  • the pipette 12 can take several positions relative to the base 34, and thus with respect to a well support 18a, 18b housed in the latter.
  • the pipette 12 can take a position in which the tips 21 of the cones 20 dipped in wells of the support 18a, 18b, and at least one position in which the tips 21 do not dip in the wells, and are at a distance from the latter to allow the handling of well supports by the user and the capture of magnetic particles in a central position of the cones 20.
  • a well support is a compartmentalized plate 18a (FIG. 5), usually called a "microplate” of the "DeepWell” type.
  • This type of plate comprises rows 60 of wells 52 in which can dip the row of pipette cones 20. Each row 60 can thus receive a particular liquid used during the extraction step implemented by the system 10. Passage from one row 60 to the other is then achieved simply by the user who puts the pipette cones 20 above the particular row 60 containing the liquid necessary for the step to be implemented.
  • the support 18b comprises for this purpose a body 64 in which is formed a row of housing 66 capable of receiving the row of pipette cone 20, and in which is housed a second magnetized piece 68 comprising one or more permanent magnets, for example a permanent magnet near each well 66.
  • the magnetized piece 68 is placed under the wells 66 or in front of their lower portions as illustrated.
  • the tips 21 of the pipette cones 20 are placed above the magnetized part 68 when said tips are immersed in the wells 66.
  • the support 18b serves as a magnetic rack removably removably tubes 69 in the housings 66 , for example, PCR elution tubes as illustrated in FIG. 6B, for purposes of, for example, subsequent transfer of the recovered extraction product thereinto.
  • the first magnetized piece 16 the function of which is to capture magnetized particles in the cones 20 in a manner described in more detail later, comprises one or more permanent magnets 72, advantageously a row of separate permanent magnets. each other by spaces 74, and still more advantageously a permanent magnet opposite each pipette cone 20 when the part 16 is completely housed in the base 34.
  • the part 16 further comprises a handle 76 for better grip by the user.
  • a housing 78 for receiving the magnetized part 16 is provided in the base 34, the housing 78 being placed so that the part 16 faces the pipette cones 20 above their point 21, and preferably facing a central zone 80 at a height greater than the well, when the tips 21 are immersed in the wells maintained in a well support. In this way, the particles are captured in a volume of the cone large enough not to form plugs in the cones.
  • the pipette holder 14 also comprises means making it possible to control the rate of rise of the support 36.
  • the rack and the gearwheel are designed so that a half-turn (180 °) of the wheel 50 makes it possible to traverse the assembly of the rack, and a counterweight 58 integrated in each of the handles 50 off-axis relative to the shaft 44.
  • These weights under their weight and the associated leverage, generate a rotational torque rotating the 44 while limiting the torque transmitted by hand by the user.
  • a substantial part of the shaft 44 is also formed of a half-cylindrical weight for the same purpose. This mechanical assistance helps lift the pipette holder, and therefore limits musculoskeletal disorders, and implements a brake that allows the user to control more accurately the rate of rise and fall of the support 36.
  • the weight 59 comprises a magnetizable material (eg steel or equivalent) and a third magnetized piece 80 (parallelepipedal or in the form of a circular arc concentric with the shaft 44) is housed in the base 34, preferably opposite the magnetized part 16 vis-à-vis the shaft 44 to not disturb the extraction.
  • the flyweight 59 of the shaft passes in front of the magnetized part 80, the rotational movement of the shaft 44 is slowed down because of the braking torque generated.
  • a stop mechanism is also advantageously provided as illustrated in FIG. 4B.
  • a wheel 84 of deformable material eg elastomer
  • a protrusion 82 for example hemispherical, is also projecting from the base 84 in front of the wheel 84.
  • the first tooth 86 meets the protrusion 82.
  • the tooth 86 bends, passes the protrusion 82, and returns to its shape .
  • the tooth 86 can then rest on the protrusion 82, the hardness of this tooth being chosen so that it does not bend under the action of the weight of the pipette 12 and the pipette holder 36.
  • the pipette is thus blocked in the high position, the user can therefore release the wheel 50.
  • the second tooth 88 of larger size (eg length and / or width) requires for its passage a much larger torque, and thus defines a stop to prevent that the pipette holder 36 does not disengage the base 34, unless the user deploys a force capable of breaking this tooth.
  • the protuberance 82 is replaced by a stop having a ball spring-loaded in a housing of the base.
  • the wheel 84 can thus be made of a hard material.
  • the action of the first tooth 86 then has the effect of pushing the ball into its housing, thus allowing the passage of the tooth 86.
  • Magnetic particles are captured sequentially in the pipette tips during suction / discharge cycles over the entire volume of sample to be treated.
  • a process for purification of viral nucleic acids using NucliSENS ⁇ chemistry namely a nucleic acid extraction using magnetic silica particles.
  • the process starts with a step 100 of preparation of the different samples and reagents necessary for the purification, followed by said purification at 102.
  • the preparation 100 consists, in 104, of mixing the biological sample comprising viruses from which it is desired to extract the nucleic acids, with a reagent for chemical lysis of viruses (eg bioMérieux's "Nuclisens miniMAG” lysis reagent, reference 200292, or bioMérieux's "Nuclisens easyMAG” lysis reagent, reference 280130), in two volumes of lysis reagent for a sample volume.
  • the mixture is then heated for 30 minutes at 56 ° C, thereby releasing the nucleic acids from the viruses in a manner known per se.
  • Magnetic silica particles eg particles having a paramagnetic core, ferromagnetic or ferrimagnetic having or not a remanence, covered with a silica shell, having the property of binding with nucleic acids, are then introduced, at 106, in the lysed sample.
  • Preparation 100 continues, at 108, by filling the microplate 18a, having wells 62 of 5mL, and PCR elution tubes 69 of 0.2ml of the magnetic rack 18b so that:
  • each well in the first row of the microplate 18a is filled with the lysed sample comprising the silica particles, hereinafter "lysed sample".
  • the total volume in each well of the first row is preferably greater than 1.5 mL due to the use of the Deepwell 5 mL microplate and volumes handled by the electronic pipette; each well 66 of the second row of the microplate 18a is filled with 1250 ⁇ of wash buffer (eg BioMérieux's "NucliSENS easyMAG Extraction Buffer No. 2" reference bMx 280131);
  • each well 66 of the third row of the microplate 18a is filled with 1250 ⁇ of wash buffer (e.g. the bioMérieux NucliSENS easyMAG Extraction Buffer # 2 reference bMx 280131);
  • wash buffer e.g. the bioMérieux NucliSENS easyMAG Extraction Buffer # 2 reference bMx 280131;
  • each PCR elution tube 69 housed in the magnetic rack 18b is filled with a volume of 100 elution buffer (e.g. the "NucliSENS easyMAG Extraction Buffer No. 3" from bioMérieux, reference 280132).
  • 100 elution buffer e.g. the "NucliSENS easyMAG Extraction Buffer No. 3" from bioMérieux, reference 280132.
  • Extraction 102 begins with homogenization of the lysed sample. To do this, the magnetized part 16 is not placed in the base 34 and therefore does not interfere with the cones 20. The user rotates one of the wheels 50 so as to immerse the tips 21 of the cones 20 in the row of plate well 18a comprising the lysed sample. Then, using the interface 24 of the pipette 12, it selects a first pipetting protocol comprising at least one suction / discharge phase of the lysed sample in the cones 20, and starts the selected protocol. These phases (e.g. two in number) each comprise at least one suction / discharge cycle (e.g. five cycles) followed by a waiting time of several minutes, for example 5 minutes. For the purposes of the invention, a suction and discharge cycle consists of filling at least three quarters, for example completely, the cones and emptying them completely, unless otherwise specified by the program.
  • a suction and discharge cycle consists of filling at least three quarters, for example completely, the cones and emptying
  • the cones 20 are empty and their tips 21 dive into the wells containing the lysed sample.
  • the purification 102 is continued by the capture, at 112, of the silica particles of the lysed sample on the inner wall of the cones 20.
  • the user places the magnetized part 16 in the housing 78 of the base 34, selects using the interface 24 of the pipette 12 a second pipetting protocol then starts the selected protocol.
  • the second protocol comprises a plurality of suction cycles / waiting / repression, eg a dozen cycles, a suction being separated from a discharge of a few seconds, eg ten seconds.
  • a part of the particles contained in the lysed sample is captured on the wall of the pipette cones by virtue of the magnetic field produced by the magnetized part 16.
  • the magnetic particles, and thus also their bound nucleic acids, are thus captured in the form of pellets of particles 100 in front of the magnetized part 16, and preferably on a central zone halfway up the cones 20, as illustrated in FIG. FIG. 9.
  • the user raises the pipette holder 14 (resp., raises the pipette 12) so as to release the plate 18a from the cones 20, aligns the second row of the plate 18a with the cone row 20 and then places the pipette holder (resp. the pipette) so as to soak the tips 21 of the cones in the wells of the plate 18a.
  • the user selects, using the interface 24, a third pipetting protocol comprising at least one suction / discharge phase of the lysed sample in the cones 20, then initiates the selected protocol.
  • the third protocol is for example identical to the first protocol. The repeated passage of the washing buffer on the pellets of particles thus makes it possible to wash the latter.
  • This washing step is advantageously completed, or implemented in conjunction with, a modulation of the magnetic field capturing the particles on the cones.
  • the user raises and lowers the pipette 12, which has the effect of moving the pellets of particles on the cones, or the magnetized part 16 comprises a train of permanent magnets and the user slides in a reciprocating the magnetic part 16 of its housing 78, so that the intensity and the magnetic field lines capturing the pellets vary, while retaining the particles captured on the cones.
  • the modulation of the magnetic field has the effect of rearranging the pellets during washing, and increase the efficiency thereof.
  • a second wash is then implemented at 116 using the washing buffer of the third row of the plate 18a. For example, the cones are completely emptied of the first wash buffer, then a second wash identical to the first wash is performed.
  • a migration step 118 of the pellets of particles 200 in the tips 21 of the cones 20 is implemented.
  • the cones 20 are preferably filled with the second washing buffer to facilitate the sliding of the caps 200 and remain aligned with the second row of the plate 18a.
  • the user then rotates one of the wheels 50 to raise the pipette 12.
  • the magnetized part 16 being secured to the base 34, the caps therefore remain immobile relative to the latter and migrate to the tips 21 by sliding on the walls of the cones 20 as the pipette rises.
  • one of the washing phases or an additional washing phase, consists in removing the magnetized part so as to release the magnetic particles and carry out a washing while stirring the particles in the washing solution by suction and discharge cycles. .
  • the particles are then captured again by repositioning the magnetized part and carrying out suction and discharge cycles as previously described.
  • the purification 102 ends with a step 120 of transfer in the PCR elution tubes 69 of the magnetic particles present in the tips 21 of the cones 20.
  • the user lifts the pipette holder 14, removes the plates 18a,
  • the magnetic rack 18b is placed in the housing 56 so as to align the PCR tubes 69 with the row of cones 20, the pipette holder 14 is rested and the magnetized piece 16 is removed from the base 14 in order to release the magnetic particles captured from the cones.
  • the user selects, using the interface 24, a fourth pipetting protocol, then launches the selected protocol.
  • a first variant of this protocol consists of suction and discharge cycles of the elution buffer in the tips 21 of the cones 20, which allows the resuspension of the magnetic particles by breaking the pellets of particles.
  • the frequency chosen for the cycles allows, at each discharge in the tubes 69, a portion of the magnetic particles to be captured in the tubes 69 by the magnetic field of the magnetized piece 68 housed in the rack 64. These cycles make it possible to "rinse" the tips 21 to recover particles adhering to the walls of the cones.
  • suction and discharge cycles are first implemented at a higher frequency so as to stir more vigorously the buffer and the particles, and thus to obtain an accelerated homogenization facilitating the transfer to the elution tubes 69.
  • the transfer step ends with the complete discharge of the elution buffer into the tubes 69.
  • the magnetic particles are then permanently separated from the buffer Elution, as illustrated in Figure 11.
  • the user can thus recover the tubes 69 for further processing, in particular the elution of nucleic acids by heating, in a manner known per se.
  • the magnetized piece 16 is housed in the base 34. Also, when the user wishes to advance the plate 18a, it can raise the pipette high enough to carry out this operation. This causes, as for the migration of particles to the tips, the displacement of the caps 200 on the walls of the cones 20, which has the advantage of "rearrange" the caps that can roll on themselves. The effectiveness of the washing is thus reinforced. However, this implies that the user is careful never to go up the pipette too much to avoid the pellets coming out of the cones. To do this, the user can for example lift or tilt the pipette holder to keep the caps away from the openings of the cones.
  • a second embodiment of the pipette holder according to the invention allows the handling of the plates 18a, 18b by raising only the pipette, and therefore avoiding lifting the pipette holder 14, while ensuring that the pellets of particles remain at a distance. tips 21 of the cones.
  • This second embodiment differs from the first embodiment by the means for receiving the workpiece.
  • the base 34 comprises the housing 78 for the insertion and removal of the magnetized part 16 as previously described and the housing 78 is open in its upper part 130 to also allow insertion. and the removal of the piece 16 vertically in the housing 78.
  • the pipette holder 36 further comprises means for fixing the magnetized piece 16 in line with the open housing 78, in particular one or more pads 132 of magnetizable material (eg steel) attached to a rear wall 134 of the movable pipette holder 36 (Fig. 12C).
  • the magnetized piece 16 is integral with the movable support 36, and remains opposite the cones 20 when the user moves up and down the pipette (in particular during the washing phases), as illustrated in FIGS. 12B to 13 A.
  • the user disconnects the magnetized piece 16 from the pipette holder 36, by simply pressing down on the handle 78 of the piece 16 and raises the pipette 12.
  • the magnetized part 16 is detached from the studs 134, thus remains in the housing 78 of the base and is thus integral with the base 34, inducing the migration of the caps 200 in the tips 21 of the cones as previously described ( Figures 13A and 13B).
  • the user replaces the plate 18a by the rack 18b equipped with PCR elution tubes, removes the magnetized part 16 and down the pipette ( Figure 13C, tubes 69 not shown).
  • the pipette holder 36 may include a housing similar to the housing 78 of the base in which the user slides the magnetized part including the wash phases.
  • a particular extraction process has been described. The present invention, however, applies to any type of capture of magnetic particles and to any type of pipetting sequence.
  • a pipette having 8 channels of a particular volume has been described.
  • the pipette may comprise any number of channels of any volume depending on the intended application.
  • two extraction systems can be coupled, as illustrated in FIG. 14.
  • the rotation handles 50 can be engaged so that two extractions can be conducted simultaneously, the user going up and down the pipettes 12 at the same time.
  • the pipettes can also be synchronized, a pipette controlling for example the other pipette.
  • the pipette is integrated with a controller that includes programmable pipette up and down and magnet movement (or electromagnet activation / deactivation) mechanisms.
  • a controller that includes programmable pipette up and down and magnet movement (or electromagnet activation / deactivation) mechanisms.
  • Two washes have been described in the wells of the second row and the third row of the microplate 18a. The number of washings can however be any.
  • a single step of capturing particles in the cones One or more particle release steps, each followed by a new capture step, may also be provided.
  • the particle capture magnetic field is deactivated by withdrawing the magnetized piece 16 from its housing, and suction / discharge cycles of a tampon are carried out in the pipette cones so as to detach the pellets. particles from the cone walls and disintegrate them.
  • suction / discharge cycles of a tampon are carried out in the pipette cones so as to detach the pellets. particles from the cone walls and disintegrate them.
  • Such a procedure takes more than 10 minutes to completely detach the caps from the cone walls with a suction / discharge frequency (full suction and discharge in the cones) of 5 cycles per minute.
  • a faster release of a pellet 200 is achieved by reciprocating, on the cap 200, the meniscus 300 that forms the pad 302 in a cone 20.
  • the suction / discharge cycle is adjusted so that the meniscus 300 makes a limited stroke 304 on either side of the base 200 in order to increase the frequency of passage of the meniscus on the base.
  • the frequency of the suction / discharge cycles is increased to further increase said passage frequency, in particular a cycle frequency greater than 2 cycles per second on the presence zone. pellet magnetic particles.
  • a full suction / discharge stirring phase in the cones is implemented (eg 8 suction / discharge cycles per minute) to complete to disaggregate the pellets and homogenize the buffer comprising the particles.
  • suction / discharge cycles in the wells of the microplates stir a larger volume, over a larger stroke, which facilitates the homogenization of the buffer.
  • nucleic acids e.g. DNA and RNA
  • This method includes a particle release phase, e.g. as previously described, followed by a buffer wash and particle recapture phase. A significant gain in time is obtained as well as improved extraction.
  • this method comprises, once the stage of lysis of the viruses carried out:
  • wash buffer eg bioMérieux's "NucliSENS easyMAG Extraction Buffer # 1" 280130.
  • Each wash comprises suction / discharge cycles of the wash buffer with particles captured on the pipette tips, and lasts at least 15 seconds, preferably between 25 seconds and 35 seconds, for example 30 seconds, and preferably less than 30 seconds. 'one minute ;
  • a third washing step in a third row of the microplate 18a filled with a washing buffer (e.gle "NucliSENS easyMAG Extraction Buffer No. 2" bioMérieux reference bMx 280131).
  • a washing buffer e.gle "NucliSENS easyMAG Extraction Buffer No. 2" bioMérieux reference bMx 280131.
  • the third washing step preferably comprising a meniscus pass phase on the pellets as described above lasts a few minutes, especially 5 minutes;
  • a second step of capturing the particles on the pipette tips eg as described above; 5. optionally a fourth washing step, the particles being captured, in a third row of the microplate 18a filled with a washing buffer (e.gle "NucliSENS easyMAG Extraction Buffer No. 2" bioMérieux reference bMx 280131);
  • a washing buffer e.gle "NucliSENS easyMAG Extraction Buffer No. 2" bioMérieux reference bMx 280131
  • extraction buffer No. 1 is a buffer promoting the capture of nucleic acids on the silica by creating bridges between the silanol groups of the silica and the phosphate groups of the nucleic acids. It comprises, for example, guanidinium thiocyanate, namely a chaotropic agent as described in the document by R. Boom et al. "Rapid and simple method for purification of nucleic acids.” Journal of Clinical Microbiology. 1989; 28 (3): 495-503;
  • the first and second washings make it possible to remove residual debris from the matrix or micro-organisms
  • the third and fourth washings make it possible to eliminate traces of GuSCN and inhibitors of a PCR type amplification which is usually subsequently put in place on the DNA / RNA captured by the magnetic particles.
  • the elution buffer included in the PCR cones makes it possible to remove all traces of washing buffer and to be in the optimal conditions for the elution step
  • the following table compares the results obtained with the device according to the invention by applying the protocol just described (first 2 wash followed by a third wash with release of the particles) in comparison with the results obtained with a device of the state. of the technique, namely the MiniMag® marketed by bioMérieux and considered as a reference device in the viral RNA extraction.
  • the protocol for the MiniMag® includes four washing steps with wash buffers (two with the "NucliSENS easyMAG Extraction Buffer # 1" buffer and two with the "NucliSENS easyMAG Extraction Buffer # 2" buffer).
  • a real-time PCR (or "q-PCR") amplification of the extracted lysate is implemented and the Ct ("cycle threshold", which quantifies a nucleic acid detection threshold in one sample) of each measured sample.
  • the samples tested in duplicate are 25-gram samples of raspberry or green onion supplemented with a solution of pure Mengo virus (corresponding to 500 copies of the genome per gram) or diluted to 1/10 th .
  • Invention (Ct value)
  • MiniMag ® Ct value
  • the extraction of the viral RNA according to the invention gives similar results with those obtained using the MiniMag®.
  • tests were conducted with different batches of magnetic silica particles of various quality. It has been found that the extraction according to the invention is surprisingly very robust vis-à-vis the quality of said particles.
  • tests were carried out on the same samples with a batch of particles of lesser performance, the extraction not having the release / washing / recapture step as described above. In this case, the extraction rate was lower. Using the preferred method described above with the defective particles, results similar to those of the previous table were obtained.
  • nucleic acids eg RNA and / or DNA from a lysis performed prior to the capture / washing / migration and transfer phases.
  • the invention is also applicable to the capture of microorganisms (eg bacteria, fungi, yeasts) using magnetic particles whose surface is functionalized to capture microorganisms (eg coated with phage proteins or polycations capable of such capture in a manner known per se).
  • the magnetic particles with their captured microorganisms are transferred into tubes for subsequent lysis, for example mechanical.
  • the lysate obtained can be directly treated, for example a polymerase chain reaction amplification (eg q-PCR quantitative PCR), or be purified according to the nucleic acid extraction method described. previously.
  • the invention is particularly suitable for microbial sample preparation for PCR.
  • the sample on which the capture of particles in the pipette cones is carried out can be of very large volume (eg several milliliters) whereas the final volume of the tubes in which the particles are transferred can be of very small volume (eg less than or equal to 200 micro liters, or even less than or equal to 100 micro liters).
  • the large volume of the sample a significant number of microorganism is captured.
  • the transition to a very small final volume has the effect of concentrating microorganisms.
  • the inventors have noted that a single capture phase from a sample of a few milliliters followed by a single washing step, is sufficient to obtain results by q-PCR from a lysis performed in a volume of 5 ml. microliters.
  • enrichment of food matrix (chicken needle) in nutrient broth was performed for 5 hours at 41.5 ° C.
  • a post-contamination with a Salmonella Derby strain is carried out at a level of 10 2 to 10 4 CFU / mL, which corresponds to concentrations that can be reached after enrichment in the presence of pathogen in the food matrix (ie concentrations for which a food lot is determined to be unsuitable for consumption).
  • concentrations for which a food lot is determined to be unsuitable for consumption concentrations for which a food lot is determined to be unsuitable for consumption.
  • two procedures are performed in duplicate, one according to the standardized capture protocol with the Gene-up® system from bioMérieux, France, and one according to the invention.
  • the Gene-up protocol consists of a step of "bead-beating" of the sample (ie mechanical disruption of the bacterial wall), taking 20 of it from a bead-beating tube containing 180 wash buffer, followed by stirring for 5 minutes on microplate shaker for bead-beating. 5 microliters of the final solution are taken and then subjected to q-PCR.
  • the process according to the invention consists of:
  • the estimated sensitivity gain is 2 log compared to the standard Gene-UP protocol.
  • the present invention addresses a problem of versatility for the use of different magnetic capture techniques (purification of nucleic acids, magnetic immuno-concentration, etc.).
  • the system according to the invention which is scalable and can be modulated, makes it possible: to carry out steps of capture / washing / elution of magnetic particles by using an autonomous system constituted by the combination of a programmable electronic pipette and a support allowing carrying out the various steps mentioned above;
  • the elution steps can be carried out in different types of tubes: 0.2 mL PCR tubes for recovering magnetic silica particles when it is It acts of capture of the nucleic acids (eg NucliSENS ⁇ chemistry) or of bead-beating tubes in the case of the recovery of magnetic particles used for the recovery of pathogens (Magnetic Immuno-Concentration).
  • the system allows the realization of pathogen capture / concentration steps on the magnetic particles and their in-situ lysis using ceramic / glass beads (e.g. CapLyse ⁇ type process).

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Abstract

The invention relates to a system for extracting analytes from a biological sample, which comprises: an electronic pipette (12) having pipette cones with a tip (21); a well holder (18a, 18b); a pipette holder (14) comprising: a base (34) into which each well holder can be inserted; a pipette bracket (36) in which the pipette (12) is inserted, movable relative to the base (34) between a first position in which the tips (21) of the cones are inserted into a well (62, 69) of the holder (18a, 18b) and at least one second position in which the tips (21) are outside said wells (62, 69); a recess (78) opposite the pipette cones (20) above the tips thereof when the pipette bracket (36) is in the first position, and facing the tips (21) of the pipette cones (21) when the pipette bracket is in the second position; and a magnetised part (16) removably inserted into the recess (78).

Description

PROCEDE ET SYSTEME EXTRACTION MAGNETIQUE DE COMPOSANTS DANS UN ECHANTILLON LIQUIDE METHOD AND SYSTEM FOR MAGNETIC EXTRACTION OF COMPONENTS IN A LIQUID SAMPLE
DOMAINE DE L'INVENTION FIELD OF THE INVENTION
L'invention a trait au domaine de l'extraction de composants contenus dans une solution en utilisant des particules magnétiques. The invention relates to the field of extracting components contained in a solution using magnetic particles.
L'invention trouve particulièrement application dans le domaine de la préparation d'échantillon biologique, notamment dans la mise en œuvre de diagnostic in vitro, par la capture d'analytes d'origine biologique (acides nucléiques, microorganismes, protéines, peptides, etc.) présents dans une solution. The invention finds particular application in the field of biological sample preparation, especially in the implementation of in vitro diagnostics, by the capture of analytes of biological origin (nucleic acids, microorganisms, proteins, peptides, etc.). ) present in a solution.
ETAT DE LA TECHNIQUE STATE OF THE ART
A l'origine développée pour l'extraction d'acides nucléiques présents dans un échantillon biologique et décrite dans le document US 5 234 809, la technologie « BOOM® » consiste à introduire dans un échantillon liquide des particules magnétiques capables de se lier avec des composants d'intérêt, puis à séparer les particules magnétiques de l'échantillon à l'aide d'un ou plusieurs aimants. Les particules ainsi capturées peuvent alors subir un traitement ultérieur par exemple pour libérer leurs composants dans une solution de récupération. En raison de l'efficacité de cette technique, de nombreux dispositifs ont été développés et commercialisés, notamment pour l'ADN et TARN, ...), tant des dispositifs manuels (par exemple le NucliSENS-miniMAG® du demandeur) que des dispositifs automatisés (NucliSENS-easyMAG® du demandeur). Ces dispositifs automatisés souffrent cependant de diverses limitations. Originally developed for the extraction of nucleic acids present in a biological sample and described in document US Pat. No. 5,234,809, the "BOOM®" technology consists in introducing into a liquid sample magnetic particles capable of binding with components of interest, then to separate the magnetic particles from the sample using one or more magnets. The particles thus captured can then undergo further processing, for example to release their components in a recovery solution. Because of the efficiency of this technique, many devices have been developed and marketed, especially for DNA and RNA, ...), both manual devices (for example the applicant's NucliSENS-miniMAG®) and devices automated systems (NucliSENS-easyMAG® of the applicant). These automated devices, however, suffer from various limitations.
Une première limitation concerne leur polyvalence et leur encombrement. En effet, ces dispositifs sont le plus souvent des automates lourds et encombrants qui sont conçus pour mettre en œuvre une séquence de traitements non modifiable par l'utilisateur. Un automate est ainsi conçu pour un seul type d'extraction, par exemple conçu pour la purification d'acides nucléiques mais incapable de mettre en œuvre une immuno-concentration magnétique. A first limitation concerns their versatility and their size. Indeed, these devices are most often heavy and bulky automata that are designed to implement a non-modifiable treatment sequence by the user. An automaton is thus designed for a single type of extraction, for example designed for the purification of nucleic acids but unable to implement a magnetic immuno-concentration.
Une deuxième limitation concerne les circuits d'injection et d'aspiration des différents liquides utilisés lors de l'extraction. Le nombre de liquides étant important, ceci implique des circuits également nombreux et/ou complexes. De plus, en raison de possibles contaminations, ces circuits d'injection/aspiration doivent être régulièrement nettoyés, ce qui implique la mise hors service des dispositifs. Une troisième limitation concerne les opérations de brassage qui sont mises en œuvre pour obtenir l'homogénéité de l'échantillon comprenant les particules magnétiques avant leur capture, pour maximiser la capture par ces dernières des analytes d'intérêt ou pour laver efficacement les particules magnétiques. Ce type de brassage nécessite usuellement des mécanismes complexes, par exemple à base d'aimants mobiles qui mettent en mouvement les particules magnétiques. A second limitation relates to the injection and suction circuits of the different liquids used during the extraction. The number of liquids being important, this involves circuits that are also numerous and / or complex. In addition, due to possible contamination, these injection / suction circuits must be regularly cleaned, which involves the disabling of the devices. A third limitation relates to the brewing operations that are implemented to obtain the homogeneity of the sample comprising the magnetic particles before capture, to maximize the capture by them of the analytes of interest or to effectively wash the magnetic particles. This type of mixing usually requires complex mechanisms, for example based on mobile magnets that set the magnetic particles in motion.
La quatrième limitation concerne les différents liquides utilisés lors de l'extraction. Usuellement, les étapes mises en œuvre pour l'extraction sont réalisées dans un ou à partir d'un seul récipient. De fait, ce récipient fixe un volume identique pour tous les liquides en jeu (e.g. l'échantillon, les différentes solutions de lavage, la solution d'élution, etc .), ce qui limite l'efficacité globale du processus d'extraction. En effet, certains traitements (e.g. le lavage) nécessitent de grands volumes pour être pleinement efficaces alors que d'autres traitements se contentent d'un petit volume de liquide (e.g. l'élution). The fourth limitation concerns the different liquids used during extraction. Usually, the steps implemented for the extraction are carried out in one or from a single container. In fact, this container sets an identical volume for all the liquids involved (e.g. the sample, the different washing solutions, the elution solution, etc.), which limits the overall efficiency of the extraction process. Indeed, some treatments (e.g. washing) require large volumes to be fully effective while other treatments are content with a small volume of liquid (e.g., elution).
EXPOSE DE L'INVENTION SUMMARY OF THE INVENTION
Le but de la présente invention est de proposer un procédé d'extraction de composants dans un échantillon liquide à l'aide de particules magnétiques qui offre une grande liberté dans le choix des volumes liquides, en particulier jusqu'à 10 ml, utilisés lors de l'extraction. The object of the present invention is to provide a method of extracting components in a liquid sample using magnetic particles which offers a great freedom in the choice of liquid volumes, in particular up to 10 ml, used during extraction.
A cet effet, l'invention a pour objet un procédé d'extraction de composants contenus dans un échantillon biologique sous forme liquide, lesdits composants étant aptes à se fixer sur des particules magnétiques, le procédé comprenant : For this purpose, the subject of the invention is a process for extracting components contained in a biological sample in liquid form, said components being capable of being fixed on magnetic particles, the process comprising:
- une phase de mélange de l'échantillon avec les particules magnétiques;  a phase of mixing the sample with the magnetic particles;
- une phase d'aspiration du mélange depuis un puits dans un cône de pipette tubulaire comprenant une pointe destinée au pipetage de liquide;  a suction phase of the mixture from a well in a tubular pipette cone comprising a tip intended for liquid pipetting;
- une phase de capture des particules magnétiques sur une paroi interne du cône de pipette : o en appliquant un premier champ magnétique au cône de pipette, ledit champ étant apte à attirer et maintenir les particules magnétiques dans une zone prédéterminée du cône de pipette, dite de « capture », au-dessus de la pointe de celui-ci ;  a phase of capture of the magnetic particles on an inner wall of the pipette cone: by applying a first magnetic field to the pipette cone, said field being able to attract and hold the magnetic particles in a predetermined zone of the pipette cone, so-called of "capture", above the tip of it;
o et en appliquant au moins un cycle d'aspiration et de refoulement du mélange contenu dans le cône de pipette dans un puits;  o and applying at least one suction and discharge cycle of the mixture contained in the pipette cone in a well;
- au moins une phase de lavage des particules capturées sur la paroi interne du cône de pipette en :  at least one washing phase of the particles captured on the inner wall of the pipette cone in:
o refoulant le mélange contenu dans le cône de pipette ; et o en appliquant depuis un puits contenant une solution de lavage au moins un cycle d'aspiration et de refoulement de la solution de lavage dans le cône de pipette ; o driving back the mixture contained in the pipette cone; and o applying from a well containing a washing solution at least one suction and discharge cycle of the washing solution into the pipette cone;
- une phase de migration des particules magnétiques sur la paroi interne du cône, depuis la zone de capture jusqu'à la pointe du cône de pipette, en réalisant un déplacement relatif du cône de la pipette par rapport au premier champ magnétique ;  a phase of migration of the magnetic particles on the inner wall of the cone, from the capture zone to the tip of the pipette cone, realizing a relative displacement of the cone of the pipette with respect to the first magnetic field;
- et une phase de transfert desdites particules magnétiques ayant migré dans la pointe du cône de pipette dans un puits de récupération contenant une solution.  and a transfer phase of said magnetic particles having migrated into the tip of the pipette cone in a recovery well containing a solution.
En d'autres termes, l'invention tire avantage d'un cône de pipette dans lequel des cycles d'aspiration et de refoulement peuvent être réalisés en trempant sa pointe dans un puits. Grâce à de tels cycles, il est possible de capturer la totalité des particules présentes dans un échantillon de volume très supérieur à celui du volume, i.e. le cône, dans lequel l'extraction est réalisée. Il est même possible de faire passer dans le cône un volume cumulé de liquide bien supérieur au volume de l'échantillon lui-même, en réglant le nombre de cycles d'aspiration et de refoulement. De la même manière, le volume de la ou des solution(s) de lavage utilisée(s) le cas échéant lors de l'extraction peut être très supérieur au volume du cône. La phase de migration permet quant à elle de localiser les particules magnétiques dans une portion du cône, la pointe, qui peut tremper dans un puits de volume très réduit. Le volume de la solution de récupération peut donc être faible si nécessaire. Le volume de la solution de récupération est ainsi indépendant du volume de l'échantillon et du volume du cône de pipette dans lequel est réalisée l'extraction. Grâce à l'invention, il est par conséquent possible d'optimiser chaque volume de liquide utilisé, et ainsi optimiser l'extraction. In other words, the invention takes advantage of a pipette cone in which suction and discharge cycles can be performed by dipping its tip in a well. By means of such cycles, it is possible to capture all the particles present in a sample of a volume much larger than that of the volume, i.e. the cone, in which the extraction is carried out. It is even possible to pass in the cone a cumulative volume of liquid well above the volume of the sample itself, by adjusting the number of suction and discharge cycles. In the same way, the volume of the washing solution (s) used (s) if necessary during the extraction can be much greater than the volume of the cone. The migration phase makes it possible to locate the magnetic particles in a portion of the cone, the tip, which can soak in a well of very small volume. The volume of the recovery solution may therefore be small if necessary. The volume of the recovery solution is thus independent of the volume of the sample and the volume of the pipette cone in which the extraction is carried out. Thanks to the invention, it is therefore possible to optimize each volume of liquid used, and thus optimize extraction.
De plus, en raison de la géométrie des cônes en forme de tube et des cycles d'aspiration refoulement, il est obtenu un brassage efficace de l'échantillon dans les cônes, brassage par exemple mis en œuvre avant la capture, ainsi qu'un lavage efficace, et ce sans faire appel à des mécanismes de type aimants mobiles. En outre, les inventeurs ont noté qu'un lavage efficace est obtenu dans le cône alors même que les particules sont capturées sur la paroi du cône de pipette. Un grand volume de solution de lavage peut être utilisé, augmentant encore l'efficacité du lavage. De fait, toutes les étapes de l'extraction (brassage, capture, lavage, transfert dans une solution de récupération) peuvent être réalisées dans le cône de pipette. In addition, because of the geometry of the tube-shaped cones and the discharge suction cycles, it is obtained an efficient mixing of the sample in the cones, stirring for example implemented before capture, as well as efficient washing, without using mobile magnets type mechanisms. In addition, the inventors have noted that effective washing is achieved in the cone even as the particles are captured on the wall of the pipette cone. A large volume of wash solution can be used, further increasing wash efficiency. In fact, all the steps of extraction (stirring, capture, washing, transfer into a recovery solution) can be performed in the pipette cone.
Selon un mode de réalisation, le déplacement du premier champ magnétique consiste à déplacer le cône de pipette parallèlement à un axe longitudinal dudit cône, et à conserver constant le premier champ magnétique, l'axe longitudinal du cône de pipette restant à égale distance du premier champ magnétique lors du déplacement du cône de pipette. En d'autres termes, la migration des particules peut être mise en œuvre de manière simple en déplaçant le cône de pipette par rapport, par exemple, un aimant permanent. According to one embodiment, the displacement of the first magnetic field consists in moving the pipette cone parallel to a longitudinal axis of said cone, and in keeping the first magnetic field constant, the longitudinal axis of the pipette cone remaining equidistant from the first one; magnetic field when moving the pipette cone. In other words, the migration of the particles can be carried out in a simple manner by moving the pipette cone relative to, for example, a permanent magnet.
Selon un mode de réalisation, la phase de transfert comprend : According to one embodiment, the transfer phase comprises:
le placement de la pointe du cône de pipette dans le puits de récupération ;  placing the tip of the pipette cone in the recovery well;
et l'application d'un second champ magnétique depuis le fond du puits de récupération de manière à faire migrer dans le puits de récupération les particules magnétiques contenues dans la pointe du cône de pipette. En particulier, le second champ magnétique est produit par un aimant positionné partiellement ou entièrement sous la pointe du cône de pipette. Le premier champ magnétique appliqué au cône de pipette est désactivé lors de l'application du second champ magnétique.  and applying a second magnetic field from the bottom of the recovery well so as to migrate in the recovery well the magnetic particles contained in the tip of the pipette cone. In particular, the second magnetic field is produced by a magnet positioned partially or wholly under the tip of the pipette cone. The first magnetic field applied to the pipette cone is disabled upon application of the second magnetic field.
En d'autres termes, le second champ magnétique permet d'attirer simplement les particules dans le puits de récupération, ce qui augmente la vitesse de récupération des particules magnétiques dans le puits de récupération, ainsi que le nombre de particules récupérées. De plus, le second champ magnétique capture automatiquement les particules magnétiques dans le puits de récupération. Par exemple, si la solution de récupération est un éluant, les composants liés aux particules ont été libérés et un technicien peut pipeter directement la solution qui est dépourvue de particules magnétiques. In other words, the second magnetic field makes it possible to simply attract the particles into the recovery well, which increases the rate of recovery of the magnetic particles in the recovery well, as well as the number of particles recovered. In addition, the second magnetic field automatically captures the magnetic particles in the recovery well. For example, if the recovery solution is an eluent, the particle-bound components have been released and a technician can directly pipet the solution which is devoid of magnetic particles.
Selon une variante privilégiée, la phase de transfert comporte la désactivation du premier champ magnétique suivie de l'application de cycles d'aspiration et de refoulement de la solution du puits de récupération dans la pointe du cône de pipette, ladite application comprenant : According to a preferred variant, the transfer phase comprises the deactivation of the first magnetic field followed by the application of suction and discharge cycles of the solution of the recovery well in the tip of the pipette cone, said application comprising:
- une première phase d'application des cycles à une première fréquence ;  a first phase of applying the cycles at a first frequency;
- suivie d'une deuxième phase d'application des cycles à une deuxième fréquence, inférieure à la première fréquence. La première phase permet de désagréger efficacement l'amas de particules capturées sur le cône de pipette, également appelé « culot », et ainsi de remettre en suspension les particules dans la solution de récupération. La deuxième phase permet de continuer à brasser la solution tout en ne s'opposant pas à la migration des particules sous l'effet du champ magnétique. Ceci permet d'augmenter encore plus la vitesse de récupération et le nombre de particules récupérées dans le puits de récupération. De plus si la solution est un éluant, dont la fonction est de libérer les composants capturés par les particules magnétiques, ces cycles ont pour effet de brasser les particules dans Γ éluant, ce qui augmente l'efficacité de Γ éluant, en particulier lors de l'utilisation d'une solution d'élution dans le décrochage des analytes des particules magnétiques sans étape de chauffage. followed by a second phase of applying the cycles at a second frequency, lower than the first frequency. The first phase effectively breaks up the cluster of particles captured on the pipette cone, also called "pellet", and thus resuspend the particles in the recovery solution. The second phase allows to continue stirring the solution while not preventing the migration of particles under the effect of the magnetic field. This makes it possible to further increase the recovery rate and the number of particles recovered in the recovery well. Moreover, if the solution is an eluent whose function is to release the components captured by the magnetic particles, these cycles have the effect of stirring the particles in the eluent, which increases the effectiveness of the eluent, in particular when using an elution solution in the stall of magnetic particle analytes without heating step.
Selon un mode de réalisation, le procédé comprend, préalablement à la phase de capture, une phase de brassage du mélange contenu dans le cône de pipette par l'application d'au moins un cycle d'aspiration et de refoulement dudit mélange dans le cône de pipette. En raison de la géométrie du cône, de forme tubulaire, il est possible d'obtenir un grand débit volumique rapporté à la section du cône, et par conséquent un brassage efficace. De plus, il existe de grandes turbulences dans le cône naturellement générées par l'écoulement du liquide, turbulences qui augmentent l'efficacité du brassage. Avantageusement, un accessoire jetable est prévu dans le cône pour accroître cet effet. According to one embodiment, the method comprises, prior to the capture phase, a stirring phase of the mixture contained in the pipette cone by the application of at least one suction and discharge cycle of said mixture in the cone pipette. Due to the cone geometry, tubular shape, it is possible to obtain a large volume flow rate to the section of the cone, and therefore effective mixing. In addition, there is great turbulence in the cone naturally generated by the flow of liquid, turbulence that increase the efficiency of the brewing. Advantageously, a disposable accessory is provided in the cone to increase this effect.
Selon un mode de réalisation, le procédé comprend, préalablement à la phase de transfert, au moins une phase de lavage des particules capturées sur la paroi interne du cône de pipette en : - en désactivant le champ magnétique ; According to one embodiment, the method comprises, prior to the transfer phase, at least one washing phase of the particles captured on the inner wall of the pipette cone by: - deactivating the magnetic field;
en libérant les particules capturées en appliquant depuis un puits contenant une solution de lavage au moins un cycle d'aspiration et de refoulement de la solution de lavage dans le cône de pipette ;  releasing the captured particles by applying from at least one well containing a wash solution at least one suction and discharge cycle of the wash solution into the pipette cone;
en appliquant une deuxième phase de capture sur une paroi interne du cône de pipette : o en appliquant le premier champ magnétique au cône de pipette  by applying a second capture phase on an inner wall of the pipette cone: o by applying the first magnetic field to the pipette cone
o et en appliquant au moins un cycle d'aspiration et de refoulement du mélange contenu dans le cône de pipette dans le puits contenant la solution de lavage.  o and applying at least one suction and discharge cycle of the mixture contained in the pipette cone in the well containing the washing solution.
Selon un mode de réalisation, la libération des particules capturées comprend une phase d'application des cycles de manière à réaliser un va-et-vient d'un ménisque de ladite solution sur un culot de particules capturées dans le cône de pipette, ledit va-et-vient dudit ménisque étant réalisé sur une portion du cône inférieure à la longueur totale du cône de pipette. According to one embodiment, the release of the captured particles comprises a phase of applying the cycles so as to reciprocate a meniscus of said solution on a pellet of particles captured in the pipette cone. and-coming of said meniscus being made on a portion of the cone lower than the total length of the pipette cone.
Plus particulièrement, la libération des particules capturées comprend une deuxième phase d'application des cycles de manière à aspirer et refouler totalement la solution de lavage du cône. La fréquence d'application des cycles de la deuxième phase est inférieure à la fréquence d'application des cycles de la première phase. More particularly, the release of the captured particles comprises a second phase of application of the cycles so as to completely suck up and discharge the washing solution of the cone. The frequency of application of the cycles of the second phase is lower than the frequency of application of the cycles of the first phase.
Notamment, préalablement à la libération des particules capturées, le procédé comprend au moins deux phases de lavage mises en œuvre dans deux solutions de lavage distinctes. In particular, prior to the release of the captured particles, the method comprises at least two washing phases implemented in two separate washing solutions.
Ce mode de réalisation est particulièrement avantageux lorsque les composants contenus dans l'échantillon biologique sont des acides nucléiques (e.g. ADN, ARN). Selon un autre mode de réalisation, les composants contenus dans l'échantillon biologique sont microorganismes (e.g. bactéries, champignons, levures), et dans lequel le procédé comprend une unique phase de capture et une unique phase de lavage. This embodiment is particularly advantageous when the components contained in the biological sample are nucleic acids (eg DNA, RNA). According to another embodiment, the components contained in the biological sample are microorganisms (eg bacteria, fungi, yeasts), and wherein the method comprises a single capture phase and a single wash phase.
En particulier, le mélange de l'échantillon avec les particules magnétiques a un volume supérieur à 1 millilitre, et de préférence supérieur ou égal à 2 millilitres, et dans lequel le volume du puits de récupération est inférieur ou égal à 200 micro litres, et de préférence inférieur ou égal à 100 micro litres. In particular, the mixture of the sample with the magnetic particles has a volume greater than 1 milliliter, and preferably greater than or equal to 2 milliliters, and in which the volume of the recovery well is less than or equal to 200 micro liters, and preferably less than or equal to 100 micro liters.
Selon un mode de réalisation, le procédé comprend, préalablement à la phase de transfert, au moins une phase de lavage des particules capturées sur la paroi interne du cône de pipette en : en aspirant de la solution de lavage dans ledit cône de pipette ; According to one embodiment, the method comprises, prior to the transfer phase, at least one washing phase of the particles captured on the inner wall of the pipette cone by: drawing the washing solution into said pipette cone;
- puis en modulant le premier champ magnétique appliqué aux particules magnétiques pour capturer celles-ci sur la paroi interne du cône de pipette ;  - Then modulating the first magnetic field applied to the magnetic particles to capture them on the inner wall of the pipette cone;
- puis en refoulant le liquide de lavage du cône de pipette.  - Then pushing the washing liquid from the pipette cone.
En d'autres termes, la modulation du champ magnétique induit une réorganisation du culot de particules capturé sur la paroi du cône. Notamment, le culot peut changer de forme, s'étaler, glisser ou encore « rouler » sur la paroi du cône de pipette. Cette réorganisation du culot permet d'augmenter encore l'efficacité du lavage, et ce d'autant plus que cette réorganisation peut être mise en œuvre conjointement avec des cycles d'aspiration et de refoulement de la solution de lavage. En particulier, la modulation du premier champ magnétique est réalisée : In other words, the modulation of the magnetic field induces a reorganization of the particle pellet captured on the wall of the cone. In particular, the base can change shape, spread, slide or "roll" on the wall of the pipette cone. This reorganization of the base makes it possible to further increase the effectiveness of the washing, and all the more so since this reorganization can be implemented in conjunction with suction and discharge cycles of the washing solution. In particular, the modulation of the first magnetic field is carried out:
en déplaçant le cône de pipette parallèlement à un axe longitudinal dudit cône, et en conservant constant le premier champ magnétique ;  moving the pipette cone parallel to a longitudinal axis of said cone, and keeping the first magnetic field constant;
et/ou en faisant défiler des aimants espacés les uns des autres devant les particules capturées.  and / or by scrolling magnets spaced apart from each other in front of the captured particles.
En d'autres termes, la modulation est obtenue simplement, par exemple par un technicien qui fait glisser une barrette d'aimants ou par un automate qui met un mécanisme simple de translation d'une barrette d'aimants. Selon un mode de réalisation, le volume du cône de pipette est au moins dix fois supérieur au volume du puits de récupération. Selon un mode de réalisation, le volume du mélange est au moins trois fois supérieur au volume du cône de pipette. Selon un mode de réalisation, les composants appartiennent au groupe formé des acides nucléiques simple brin ou double brin (ADN et/ou ARN), des microorganismes, des protéines, et des peptides. Les composants sont constitués de tout autres types de molécules en fonction de la fonctionnalisation données aux particules magnétiques. In other words, the modulation is obtained simply, for example by a technician who slides a bar of magnets or by a PLC which puts a simple mechanism of translation of a bar of magnets. According to one embodiment, the volume of the pipette cone is at least ten times greater than the volume of the recovery well. According to one embodiment, the volume of the mixture is at least three times greater than the volume of the pipette cone. According to one embodiment, the components belong to the group consisting of single-stranded or double-stranded nucleic acids (DNA and / or RNA), microorganisms, proteins, and peptides. The components consist of all other types of molecules depending on the functionalization given to the magnetic particles.
Le but de la présente invention est également de proposer un dispositif pour la mise en œuvre du procédé venant d'être décrit, qui soit peu encombrant et simple d'utilisation par un technicien de laboratoire. A cet effet, l'invention a également pour objet un porte-pipette comprenant : The object of the present invention is also to provide a device for implementing the method just described, which is compact and easy to use by a laboratory technician. For this purpose, the invention also relates to a pipette holder comprising:
un socle ;  a pedestal ;
un évidement formé dans le socle apte à loger de manière amovible un support de puits ; un support de pipette comprenant un premier logement dans lequel est apte à être insérée, avantageusement de manière amovible, une pipette équipée d'au moins un cône de pipette tubulaire comprenant une pointe destinée au pipetage de liquide, le premier logement étant ouvert sur l'évidement du socle, le support de pipette étant mobile en translation par rapport au socle selon une direction parallèle à un axe des cônes de pipette et mobile entre une première position dans laquelle la pointe de chaque cône de pipette est logée dans un puits du support de puits et au moins une deuxième position dans laquelle ladite pointe est en dehors dudit puits;  a recess formed in the base adapted to removably accommodate a well support; a pipette holder comprising a first housing in which is insertable, advantageously removably, a pipette equipped with at least one tubular pipette cone comprising a tip for liquid pipetting, the first housing being open on the recess of the base, the pipette holder being movable in translation relative to the base in a direction parallel to an axis of the pipette cones and movable between a first position in which the tip of each pipette cone is housed in a well of the support of well and at least a second position in which said tip is outside said well;
un second logement apte à loger de manière amovible une pièce aimantée, le second logement faisant face à chacun des cônes de pipette dans une position au-dessus de la pointe de celui-ci lorsque le support de pipette est dans la première position, et le second logement fait face à la pointe du cône de pipette lorsque le support de pipette est dans la deuxième position.  a second housing adapted to releasably receive a magnetized part, the second housing facing each of the pipette tips in a position above the tip thereof when the pipette holder is in the first position, and the second housing faces the tip of the pipette cone when the pipette holder is in the second position.
En d'autres termes, le porte-pipette reçoit une pipette et le technicien met en œuvre les étapes du procédé d'extraction, en montant/descendant la pipette, notamment pour faire migrer le culot de particule dans la pointe du ou des cônes de pipette, en introduisant des puits (sous la forme de plaque, de barrette, etc) dans le socle, et en actionnant la pipette. In other words, the pipette holder receives a pipette and the technician implements the steps of the extraction process, up / down the pipette, in particular to migrate the particle pellet in the tip of the cone (s). pipette, introducing wells (in the form of plate, bar, etc.) into the base, and actuating the pipette.
Le porte-pipette, qui est peu encombrant et transportable, permet en outre la semi- automatisation du procédé d'extraction lorsqu'une pipette électronique est utilisée. Une telle pipette comprend en effet des circuits d'aspiration et de refoulement dans chaque cône de pipette l'équipant, et un circuit électronique à base de microprocesseur. Ce circuit électronique pilote les circuits d'aspiration/refoulement en fonction de consignes entrées par le technicien au moyen d'une interface équipant la pipette et/ou d'un ordinateur/tablette/smartphone connecté à la pipette (e.g. par une liaison sans fil de type bluetooth), etc. Ces consignes sont par exemple constituées d'instructions de cycles d'aspiration/refoulement et/ou d'un choix d'un protocole particulier préenregistré dans la pipette. La pipette électronique étant programmable, une grande polyvalence est en outre obtenue dans la définition du procédé d'extraction, qui peut être adapté en fonction d'une capture magnétique particulière souhaitée (e.g. : purification d'acides nucléiques, immuno- concentration magnétiques,...). Notamment, un protocole approprié à l'extraction visée peut être enregistré dans la pipette, le protocole étant défini en termes de nombre de cycles d'aspiration et de refoulement, d'enchaînement de cycles, de fréquence de cycles, de durée entre les cycles, volumes définis, etc. Un système autonome et semi-automatisé est ainsi obtenu. The pipette holder, which is compact and transportable, also allows semi-automation of the extraction process when an electronic pipette is used. Such a pipette comprises indeed suction and discharge circuits in each pipette cone equipping it, and a microprocessor-based electronic circuit. This electronic circuit controls the suction / discharge circuits according to instructions entered by the technician by means of an interface fitted to the pipette and / or a computer / tablet / smartphone connected to the pipette (eg via a wireless link Of type bluetooth), etc. These instructions consist, for example, of instructions for suction / discharge cycles and / or of a choice of a particular protocol pre-recorded in the pipette. Since the electronic pipette is programmable, great versatility is furthermore obtained in the definition of the extraction process, which can be adapted according to a particular desired magnetic capture (eg: purification of nucleic acids, magnetic immunoconcentration ,. ..). In particular, a suitable protocol for the targeted extraction can be recorded in the pipette, the protocol being defined in terms of the number of suction and discharge cycles, the sequence of cycles, the frequency of cycles, the duration between cycles. , defined volumes, etc. An autonomous and semi-automated system is thus obtained.
Enfin, les cônes de pipette sont détachables de la pipette, et donc aisément remplaçables, sans que la pipette ne soit mise hors service pendant une longue durée. Finally, the pipette tips are detachable from the pipette, and therefore easily replaceable, without the pipette is disabled for a long time.
Selon un mode de réalisation, le premier logement comprend une ouverture pour l'insertion et le retrait frontaux de la pipette dans le premier logement du support de pipette. L'insertion et le retrait frontaux de la pipette et des cônes en position minimisent le risque de toucher le porte-pipette avec les pointes des cônes, et donc le risque de contamination du porte-pipette. In one embodiment, the first housing includes an opening for the front insertion and withdrawal of the pipette into the first housing of the pipette holder. The front insertion and removal of the pipette and cones in place minimizes the risk of touching the pipette holder with cone tips, and thus the risk of contamination of the pipette holder.
Selon un mode de réalisation, le second logement est réalisé dans le socle. According to one embodiment, the second housing is made in the base.
En particulier, le support de pipette comporte un troisième logement dans lequel la pièce aimantée est apte à être logée de manière amovible pour faire face à chaque cône de pipette à une position au-dessus de la pointe dudit cône lorsque le support de pipette est dans la deuxième position. In particular, the pipette holder has a third housing in which the magnetized piece is removable to face each pipette cone at a position above the tip of said cone when the pipette holder is in the second position.
En d'autres termes, lorsque la pièce aimantée (e.g. comprenant un ou plusieurs aimants permanents) est présente dans le troisième logement, elle est solidaire des cônes de pipette et suit donc leur mouvement de translation par rapport au socle. Lors de tels mouvements, la pièce aimantée conserve donc les culots de particules magnétiques fixes dans les cônes. Le technicien peut ainsi par exemple monter la pipette pour déplacer plus aisément un support à puits dans le socle sans risquer de déplacer les culots de particules dans les cônes. In other words, when the magnetized part (e.g. comprising one or more permanent magnets) is present in the third housing, it is integral with the pipette tips and therefore follows their translational movement relative to the base. During such movements, the magnetized part thus retains the pellets of magnetic particles fixed in the cones. The technician can for example mount the pipette to more easily move a well support in the base without the risk of moving the pellets of particles in the cones.
Selon un mode particulier, le second et troisième logements communiquent, et le support de pipette comprend des moyens aptes à maintenir de manière amovible la pièce aimantée dans le troisième logement. De cette manière, le technicien peut détacher la pièce aimantée du support à pipette qui prend alors place automatiquement dans le socle en tombant dans le deuxième logement. Ce détachement a notamment lieu pour l'opération de migration des particules magnétiques dans les pointes des cônes. Selon un mode de réalisation : According to a particular mode, the second and third housings communicate, and the pipette support comprises means able to removably hold the magnetized part in the third housing. In this way, the technician can detach the magnetized piece from pipette holder which then automatically takes place in the base by falling into the second housing. This detachment in particular takes place for the migration operation of the magnetic particles in the tips of the cones. According to one embodiment:
le socle comprend au moins une roue dentée mobile en rotation ;  the base comprises at least one rotating gear wheel;
et le support de pipette comprend une crémaillère coopérant avec la roue dentée pour translater le support de pipette par rapport au socle lors de la rotation de la roue dentée. En particulier, le porte-pipette comprend un dispositif de verrouillage et de déverrouillage du support de pipette dans la première position. Notamment, le porte-pipette comprend au moins une poignée solidaire de la roue dentée pour faire tourner celle-ci et apte à se fixer de manière amovible à une poignée solidaire de la roue dentée d'un autre porte-pipette, ce qui permet donc d'augmenter le nombre de cônes de pipette pendant le procédé d'extraction.  and the pipette holder comprises a rack cooperating with the gearwheel to translate the pipette holder relative to the base during rotation of the gearwheel. In particular, the pipette holder comprises a device for locking and unlocking the pipette holder in the first position. In particular, the pipette holder comprises at least one handle secured to the toothed wheel to rotate it and adapted to be removably attached to a handle secured to the gear wheel of another pipette holder, which therefore allows to increase the number of pipette tips during the extraction process.
L'invention a également pour objet un système pour l'extraction de composants contenus dans un échantillon biologique sous forme liquide, lesdits composants étant aptes à se fixer sur des particules magnétiques, le système comprenant : The invention also relates to a system for extracting components contained in a biological sample in liquid form, said components being capable of being fixed on magnetic particles, the system comprising:
- une pipette équipée d'au moins un cône de pipette tubulaire comprenant une pointe destinée au pipetage de liquide et d'un circuit d'aspiration et de refoulement dans chaque cône de pipette ;  a pipette equipped with at least one tubular pipette cone comprising a tip intended for liquid pipetting and a suction and discharge circuit in each pipette cone;
au moins un support de puits ;  at least one well support;
un porte-pipette comprenant :  a pipette holder comprising:
o un socle ;  o a pedestal;
o un évidement formé dans le socle apte à recevoir de manière amovible chaque support de puits ;  o a recess formed in the base adapted to removably receive each well support;
o un support de pipette comprenant un premier logement dans lequel la pipette est insérée, avantageusement de manière amovible, le premier logement étant ouvert sur l'évidement du socle, le support de pipette étant mobile en translation par rapport au socle selon une direction parallèle à un axe des cônes de pipette et mobile entre une première position dans laquelle la pointe de chaque cône de pipette est logée dans un puits du support de puits et au moins une deuxième position dans laquelle ladite pointe est en dehors dudit puits;  a pipette support comprising a first housing in which the pipette is inserted, advantageously removably, the first housing being open on the recess of the base, the pipette support being movable in translation relative to the base in a direction parallel to an axis of the pipette cones and movable between a first position in which the tip of each pipette cone is housed in a well of the well holder and at least a second position in which said tip is out of said well;
o un second logement faisant face à chacun des cônes de pipette dans une position au- dessus de la pointe de celui-ci lorsque le support de pipette est dans la première position et le second logement faisant face à la pointe du cône de pipette lorsque le support de pipette est dans la deuxième position ; et  a second housing facing each of the pipette tips in a position above the tip thereof when the pipette holder is in the first position and the second housing facing the tip of the pipette cone when the pipette holder is in the second position; and
une pièce aimantée logée de manière amovible dans le second logement. Notamment, le porte-pipette est conforme au porte-pipette décrit plus haut. a magnetized piece removably housed in the second housing. In particular, the pipette holder is in accordance with the pipette holder described above.
L'invention a également pour but de proposer un support de puits pour la migration de particules magnétiques depuis des pointes de cônes de pipettes dans des puits de récupération. The invention also aims to provide a well support for the migration of magnetic particles from tips of pipette cones in recovery wells.
A cet effet, l'invention a également pour objet un support de puits, comprenant une pièce dans laquelle sont formés des évidements pour la réception des puits, et au moins un aimant faisant face à chacun des évidements formés dans ladite pièce. For this purpose, the invention also relates to a well support, comprising a part in which are formed recesses for receiving the wells, and at least one magnet facing each of the recesses formed in said part.
BRÈVE DESCRIPTION DES FIGURES BRIEF DESCRIPTION OF THE FIGURES
L'invention sera mieux comprise à la lecture de la description qui va suivre, donnée uniquement à titre d'exemple, et faite en relation avec les dessins annexés, dans lesquels des références identiques désignent des éléments identiques, et dans lesquels : The invention will be better understood on reading the following description, given solely by way of example, and with reference to the accompanying drawings, in which identical references designate identical elements, and in which:
la figure 1 est une vue en perspective d'un système d'extraction selon l'invention ;  Figure 1 is a perspective view of an extraction system according to the invention;
les figures 2A et 2B sont des vues de face et en perspective d'une pipette électronique et de ses cônes de pipettes amovibles ;  FIGS. 2A and 2B are front and perspective views of an electronic pipette and its removable pipette tips;
les figures 3A et 3B est des vues en perspective et de face d'un porte-pipette selon l'invention ;  FIGS. 3A and 3B are perspective and front views of a pipette holder according to the invention;
les figures 4A et 4B est une vue de détail en coupe du porte-pipette de la figure 3, respectivement selon les plans A-A et B-B de la figure 3B ;  FIGS. 4A and 4B are a detail sectional view of the pipette holder of FIG. 3, respectively along the planes A-A and B-B of FIG. 3B;
la figure 5 est une vue en perspective d'une plaque « deepwell » comprenant des puits ; les figures 6A et 6B sont des vues en perspective d'un portoir magnétique et de tubes d'élution PCR pouvant se loger dans le portoir ;  Figure 5 is a perspective view of a deepwell plate including wells; Figures 6A and 6B are perspective views of a magnetic rack and PCR elution tubes that can be accommodated in the rack;
la figure 7 est une vue de face d'une pièce aimantée selon l'invention ;  Figure 7 is a front view of a magnetized part according to the invention;
la figure 8 est un organigramme d'un procédé d'extraction selon l'invention ;  Figure 8 is a flow chart of an extraction process according to the invention;
la figure 9 est une photographie des cônes de pipette d'un système selon l'invention avec des culots de particules magnétiques placés environ à mi-hauteur des cônes ;  Figure 9 is a photograph of the pipette tips of a system according to the invention with pellets of magnetic particles placed about halfway up the cones;
- la figure 10 est une photographie de ces mêmes cônes avec les culots placés dans les pointes de ceux-ci ;  - Figure 10 is a photograph of these same cones with the caps placed in the tips thereof;
la figure 11 est une photographie de tubes d'élution PCR dans lesquels ont été récupérées les particules magnétiques :  FIG. 11 is a photograph of PCR elution tubes in which the magnetic particles have been recovered:
les figures 12 et 13 illustrent un second mode de réalisation du porte-pipette selon l'invention ;  Figures 12 and 13 illustrate a second embodiment of the pipette holder according to the invention;
la figure 14 est une vue en perspective de deux systèmes selon la figure 1 couplés au moyen de poignées de rotation ; la figure 15 est une vue schématique illustrant le va-et-vient d'un ménisque d'une solution sur un culot de particules pour détacher ce dernier de la paroi d'un cône de pipette . Excepté pour la figure 15, la description est réalisée en relation avec des plans et photographies à une échelle réduite d'un système réel. Figure 14 is a perspective view of two systems according to Figure 1 coupled by means of rotation handles; Figure 15 is a schematic view illustrating the reciprocation of a meniscus of a solution on a particle pellet to detach the latter from the wall of a pipette cone. Except for Figure 15, the description is made in relation to plans and photographs on a reduced scale of a real system.
DESCRIPTION DÉTAILLÉE DE L'INVENTION En se référant aux figures 1 à 7, un système 10 d'extraction (figure 1) de composants contenus dans un échantillon liquide comporte une pipette électronique 12 (figures 2), un porte-pipette 14 (figures 3) dans lequel la pipette 12 est logée, un ou plusieurs support de puits 18a, 18b (figure 5 et 6) pouvant chacun être logé dans le porte-pipette 14, et une première pièce aimantée 16 (figure 7). DETAILED DESCRIPTION OF THE INVENTION Referring to FIGS. 1 to 7, a system 10 for extracting (FIG. 1) components contained in a liquid sample comprises an electronic pipette 12 (FIG. 2), a pipette holder 14 (FIGS. ) in which the pipette 12 is housed, one or more well supports 18a, 18b (FIGS. 5 and 6) can each be housed in the pipette holder 14, and a first magnetized piece 16 (FIG. 7).
La pipette 12, qui est portable, comprend une rangée de cônes de pipettes 20, et un corps 22 sur lequel sont montés les cônes 20 (figure 2A). Dans ce corps 20, sont logés un circuit d'aspiration/refoulement de liquide dans les cônes 20 (par exemple un ensemble de pistons actionnés par un moteur électrique), et un circuit électronique de pilotage du circuit d'aspiration/refoulement. Le circuit électronique, qui comprend par exemple un microprocesseur et une ou plusieurs mémoires informatiques, est programmable, et embarque des instructions pour la mise en œuvre d'un ou plusieurs protocoles de pipetage, chaque protocole comprenant une ou plusieurs étapes. Le circuit électronique comprend également une interface homme machine 24 logée dans une poignée 26 du corps 22, l'interface comprenant un ensemble de boutons de sélection et de navigation 28 et un écran d'affichage 30 permettant la visualisation et la sélection de différents protocoles de pipetage enregistrés. L'utilisateur peut notamment programmer la pipette électronique 12, e.g. en téléchargeant dans celle-ci des instructions depuis un ordinateur connecté à la pipette 12 au travers d'une liaison sans fil, par exemple Bluetooth. L'utilisateur peut également sélectionner, via l'interface 24, un protocole préenregistré. La pipette 12 est par ailleurs apte à aspirer un volume prédéfini de liquide dans chacun des cônes 20, refouler un volume prédéfini depuis chacun des cônes, mettre en œuvre des cycles automatiques d'aspiration/refoulement de durée et de fréquence variable. Comme illustré plus particulièrement à la figure 2B, les cônes 20, de forme tubulaires et d'axe longitudinal X, sont amovibles en s 'emboîtant dans des lobes 32 faisant saillie du corps 22, ce qui permet leur remplacement. Les cônes sont par ailleurs constitués d'un matériau plastique, par exemple en polypropylène, ce qui a pour effet de les rendre « transparent » à un champ magnétique et permet la capture de particules magnétiques, comme cela sera décrit plus en détails par la suite. Enfin, chaque cône 20 présente à son extrémité ouverte de pipetage un profil effilé 21, ou « pointe ». Cette partie 21 a une section réduite dans un plan perpendiculaire à l'axe X, ce qui permet son introduction facilitée dans des récipients ou puits comme cela est connu en soi. The pipette 12, which is portable, comprises a row of pipette tips 20, and a body 22 on which the cones 20 are mounted (FIG. 2A). In this body 20 are housed a liquid suction / discharge circuit in the cones 20 (for example a set of pistons actuated by an electric motor), and an electronic control circuit of the suction / discharge circuit. The electronic circuit, which comprises for example a microprocessor and one or more computer memories, is programmable, and embeds instructions for the implementation of one or more pipetting protocols, each protocol comprising one or more steps. The electronic circuit also comprises a man-machine interface 24 housed in a handle 26 of the body 22, the interface comprising a set of selection and navigation buttons 28 and a display screen 30 allowing the display and selection of different control protocols. pipetting recorded. The user can in particular program the electronic pipette 12, eg by downloading instructions therefrom from a computer connected to the pipette 12 through a wireless link, for example Bluetooth. The user can also select, via the interface 24, a prerecorded protocol. The pipette 12 is also able to suck up a predefined volume of liquid in each of the cones 20, to discharge a predefined volume from each of the cones, to implement automatic suction / discharge cycles of variable duration and frequency. As illustrated more particularly in FIG. 2B, the cones 20, of tubular shape and of longitudinal axis X, are removable by fitting into lobes 32 protruding from the body 22, which allows their replacement. The cones are also made of a plastic material, for example polypropylene, which has the effect of making them "transparent" to a field magnetic and allows the capture of magnetic particles, as will be described in more detail later. Finally, each cone 20 has at its open pipetting end a tapered profile 21, or "tip". This portion 21 has a reduced section in a plane perpendicular to the X axis, which allows its introduction facilitated in containers or wells as is known per se.
La pipette électronique 12 est par exemple le modèle « VIAFLO II 8 canaux » commercialisé par la société ©INTEGRA Biosciences AG, Suisse, modèle dont les éléments sont décrits dans les demandes de brevets US 2009/071266, US 2009/074622, US2011076205 et US 2008/095671. The electronic pipette 12 is for example the "VIAFLO II 8-channel" model marketed by the company © INTEGRA Biosciences AG, Switzerland, the model of which elements are described in the patent applications US 2009/071266, US 2009/074622, US2011076205 and US 2008/095671.
Le porte-pipette 14 comporte quant à lui (figures 3A et 3B) un socle 34, destiné à être posé sur un plan de travail (e.g. une table ou une paillasse de laboratoire), ainsi qu'une partie mobile 36 par rapport au socle 34. La partie mobile 36, également nommée « support de pipette », comporte un logement 38 dans lequel la pipette 12 peut être insérée de manière amovible et maintenue immobile comme illustré à la figure 1. Pour la translation du support de pipette 36 par rapport au socle 34, le support 36 comporte une ou plusieurs crémaillères 40, par exemple au nombre de deux, s 'engrenant avec des roues dentées 42 montées sur un arbre 44 mobile en rotation et logé dans le socle 34. Une ou plusieurs tiges 46 sont en outre fixées au support 36 (resp. dans le socle 34) et coulissent dans des orifices du socle 34 (resp. dans le support 36), afin de guider le support 36 dans son mouvement de translation. Une ou deux poignées de rotation 50 sont en outre fixées au bout de l'arbre 44 afin de permettre à l'utilisateur de facilement tourner ce dernier selon les flèches 52 (figure 1) et donc faire monter et descendre le support de pipette 36 tel qu'illustré par les flèches 54 (figure 1). Le mouvement de translation du support de pipette 36 par rapport au socle 34 est donc parallèle à l'axe X des cônes 20 lorsque la pipette 12 est placée dans le logement 38 du support 36, et donc parallèle à la direction de la gravité lorsque le socle 34 est posé sur un plan de travail horizontal, de sorte que le support 36 « monte » ou « descend». Le socle 34 est ouvert sur sa face avant 56 pour permettre l'introduction et le retrait des supports de puits 18a, 18b, définissant ainsi un logement pour ces derniers. Ce logement est ouvert sur sa partie supérieure pour permettre aux cônes 20 de la pipette 12 d'atteindre lesdits supports de puits lorsque la pipette descend. Ainsi, comme décrit plus en détail ci-après, la pipette 12 peut prendre plusieurs positions par rapport au socle 34, et donc par rapport à un support de puits 18a, 18b logé dans ce dernier. En particulier, la pipette 12 peut prendre une position dans laquelle les pointes 21 des cônes 20 trempent dans des puits du support 18a, 18b, et au moins une position dans laquelle les pointes 21 ne trempent pas dans les puits, et sont à distance de ces derniers de manière à permettre la manipulation des supports de puits par l'utilisateur et la capture de particules magnétiques dans une position centrale des cônes 20. The pipette holder 14 comprises meanwhile (FIGS. 3A and 3B) a base 34 intended to be placed on a work surface (eg a laboratory table or bench) and a mobile part 36 with respect to the base. 34. The mobile part 36, also called "pipette holder", comprises a housing 38 in which the pipette 12 can be removably inserted and kept stationary as illustrated in FIG. 1. For the translation of the pipette holder 36 relative to at the base 34, the support 36 comprises one or more racks 40, for example two in number, meshing with gear wheels 42 mounted on a shaft 44 rotatable and housed in the base 34. One or more rods 46 are furthermore fixed to the support 36 (or in the base 34) and slide in orifices of the base 34 (or in the support 36), in order to guide the support 36 in its translational movement. One or two rotational handles 50 are further attached to the end of the shaft 44 to allow the user to easily turn the latter according to the arrows 52 (Figure 1) and thus raise and lower the pipette holder 36 as as illustrated by the arrows 54 (Figure 1). The translational movement of the pipette holder 36 relative to the base 34 is therefore parallel to the axis X of the cones 20 when the pipette 12 is placed in the housing 38 of the support 36, and therefore parallel to the direction of gravity when the base 34 is placed on a horizontal work plane, so that the support 36 "up" or "down". The base 34 is open on its front face 56 to allow the introduction and removal of the well supports 18a, 18b, thus defining a housing for the latter. This housing is open on its upper part to allow the cones 20 of the pipette 12 to reach said well supports when the pipette goes down. Thus, as described in more detail below, the pipette 12 can take several positions relative to the base 34, and thus with respect to a well support 18a, 18b housed in the latter. In particular, the pipette 12 can take a position in which the tips 21 of the cones 20 dipped in wells of the support 18a, 18b, and at least one position in which the tips 21 do not dip in the wells, and are at a distance from the latter to allow the handling of well supports by the user and the capture of magnetic particles in a central position of the cones 20.
En se référant aux figures 5 et 6, les supports de puits peuvent prendre plusieurs formes en fonction de l'extraction souhaitée. Notamment, un support de puits est une plaque 18a compartimentée (figure 5), usuellement nommée « microplaque » de type « DeepWell ». Ce type de plaque comporte des rangées 60 de puits 52 dans lesquelles peut plonger la rangée de cônes de pipette 20. Chaque rangée 60 peut ainsi recevoir un liquide particulier utilisé lors de l'étape d'extraction mise en œuvre par le système 10. Le passage d'une rangée 60 à l'autre est alors réalisé simplement par l'utilisateur qui met à l'aplomb des cônes de pipettes 20 la rangée 60 particulière contenant le liquide nécessaire à l'étape devant être mise en œuvre. Referring to FIGS. 5 and 6, the well supports can take a number of forms depending on the desired extraction. In particular, a well support is a compartmentalized plate 18a (FIG. 5), usually called a "microplate" of the "DeepWell" type. This type of plate comprises rows 60 of wells 52 in which can dip the row of pipette cones 20. Each row 60 can thus receive a particular liquid used during the extraction step implemented by the system 10. Passage from one row 60 to the other is then achieved simply by the user who puts the pipette cones 20 above the particular row 60 containing the liquid necessary for the step to be implemented.
Un autre support de puits, décrit à la figure 6A, est aimanté et spécifiquement conçu pour la migration de particules magnétiques depuis les pointes 21 des cônes de pipettes 20 dans des puits. Le support 18b comprend à cet effet un corps 64 dans lequel est formée une rangée de de logement 66 pouvant recevoir la rangée de cône de pipette 20, et dans lequel est logée une seconde pièce aimantée 68 comprenant un ou plusieurs aimants permanents, par exemple un aimant permanent à proximité de chacun des puits 66. La pièce aimantée 68 est placée sous les puits 66 ou en face de leur portions inférieures comme illustré. Ainsi, les pointes 21 des cônes de pipettes 20 sont placées au-dessus de la pièce aimantée 68 lorsque lesdites pointes sont plongées dans les puits 66. Enfin, le support 18b sert de portoir magnétique revevant de manière amovible des tubes 69 dans les logements 66, par exemple des tubes d'élution PCR tels qu'illustrés à la figure 6B, à des fins par exemple de transfert ultérieur du produit de l'extraction récupéré dans ceux-ci. Another well support, depicted in FIG. 6A, is magnetized and specifically designed for the migration of magnetic particles from the tips 21 of the pipette tips 20 into wells. The support 18b comprises for this purpose a body 64 in which is formed a row of housing 66 capable of receiving the row of pipette cone 20, and in which is housed a second magnetized piece 68 comprising one or more permanent magnets, for example a permanent magnet near each well 66. The magnetized piece 68 is placed under the wells 66 or in front of their lower portions as illustrated. Thus, the tips 21 of the pipette cones 20 are placed above the magnetized part 68 when said tips are immersed in the wells 66. Finally, the support 18b serves as a magnetic rack removably removably tubes 69 in the housings 66 , for example, PCR elution tubes as illustrated in FIG. 6B, for purposes of, for example, subsequent transfer of the recovered extraction product thereinto.
La première pièce aimantée 16, dont la fonction est de capturer des particules aimantées dans les cônes 20 d'une manière décrite plus en détail par la suite, comprend quant à elle un ou plusieurs aimants permanents 72, avantageusement une rangée d'aimants permanents séparés les uns des autres par des espaces 74, et encore plus avantageusement un aimant permanent en face de chaque cône de pipette 20 lorsque la pièce 16 est entièrement logée dans le socle 34. La pièce 16 comprend en outre une poignée 76 pour une meilleure préhension par l'utilisateur. The first magnetized piece 16, the function of which is to capture magnetized particles in the cones 20 in a manner described in more detail later, comprises one or more permanent magnets 72, advantageously a row of separate permanent magnets. each other by spaces 74, and still more advantageously a permanent magnet opposite each pipette cone 20 when the part 16 is completely housed in the base 34. The part 16 further comprises a handle 76 for better grip by the user.
Un logement 78 pour la réception de la pièce aimantée 16 est prévu dans le socle 34, le logement 78 étant placé de manière à ce que la pièce 16 soit en face des cônes de pipette 20 au-dessus de leur pointe 21, et de préférence face à une zone centrale 80 à une hauteur supérieure au puits , lorsque les pointes 21 plongent dans les puits maintenu dans un support de puits. De cette manière, les particules sont capturées dans un volume du cône suffisamment grand pour ne pas former de bouchons dans les cônes. Le porte-pipette 14 comporte également des moyens permettant de maîtriser la vitesse de remontée du support 36. En particulier, la crémaillère et la roue dentée sont conçus pour qu'un demi-tour (180°) de la roue 50 permette de parcourir l'ensemble de la crémaillère, et une masselotte 58 intégrée dans chacune des poignées 50 de manière désaxée par rapport à l'arbre 44. Ces masselottes, sous leur poids et l'effet de levier associé, génèrent un couple de rotation mettant en rotation l'arbre 44 tout en limitant le couple transmis à la main par l'utilisateur. De manière avantageuse, comme illustré à la figure 4A, une partie substantielle de l'arbre 44 est formé également d'une masselotte demi- cylindrique dans le même but. Cette assistance mécanique aide à soulever le support-de pipette, et limite donc les troubles musculo-squelettiques, et met en œuvre un frein qui permet à l'utilisateur de contrôler avec plus de précision la vitesse de montée et de descente du support 36. A housing 78 for receiving the magnetized part 16 is provided in the base 34, the housing 78 being placed so that the part 16 faces the pipette cones 20 above their point 21, and preferably facing a central zone 80 at a height greater than the well, when the tips 21 are immersed in the wells maintained in a well support. In this way, the particles are captured in a volume of the cone large enough not to form plugs in the cones. The pipette holder 14 also comprises means making it possible to control the rate of rise of the support 36. In particular, the rack and the gearwheel are designed so that a half-turn (180 °) of the wheel 50 makes it possible to traverse the assembly of the rack, and a counterweight 58 integrated in each of the handles 50 off-axis relative to the shaft 44. These weights, under their weight and the associated leverage, generate a rotational torque rotating the 44 while limiting the torque transmitted by hand by the user. Advantageously, as illustrated in FIG. 4A, a substantial part of the shaft 44 is also formed of a half-cylindrical weight for the same purpose. This mechanical assistance helps lift the pipette holder, and therefore limits musculoskeletal disorders, and implements a brake that allows the user to control more accurately the rate of rise and fall of the support 36.
D'autres mécanismes de contrôle de la vitesse du support 36 peuvent être prévus, notamment un freinage magnétique. Par exemple, en se référant à la figure 4A, la masselotte 59 comprend un matériau aimantable (e.g. acier ou équivalent) et une troisième pièce aimantée 80 (parallélépipédique ou en forme d'arc de cercle concentrique à l'arbre 44) est logée dans le socle 34, de préférence à l'opposé de la pièce aimantée 16 vis-à-vis de l'arbre 44 pour ne pas perturber l'extraction. Lorsque la masselotte 59 de l'arbre passe devant la pièce aimantée 80, le mouvement de rotation de l'arbre 44 est ralenti en raison du couple de freinage engendré. Ceci permet d'une part de compenser l'effort pour soulever la pipette lors de la montée (en jouant le rôle d'assistance pour l'utilisateur), et d'autre part de maîtriser la vitesse de remontée de la pipette lorsque l'on veut faire descendre les particules magnétiques en bas des cônes de pipette, comme cela sera décrit ci-dessous. Other mechanisms for controlling the speed of the support 36 may be provided, in particular magnetic braking. For example, with reference to FIG. 4A, the weight 59 comprises a magnetizable material (eg steel or equivalent) and a third magnetized piece 80 (parallelepipedal or in the form of a circular arc concentric with the shaft 44) is housed in the base 34, preferably opposite the magnetized part 16 vis-à-vis the shaft 44 to not disturb the extraction. When the flyweight 59 of the shaft passes in front of the magnetized part 80, the rotational movement of the shaft 44 is slowed down because of the braking torque generated. This allows on the one hand to compensate for the effort to lift the pipette during the climb (playing the role of assistance for the user), and on the other hand to control the speed of rise of the pipette when the it is desired to lower the magnetic particles down the pipette tips, as will be described below.
Un mécanisme de butée est également avantageusement prévu comme illustré à la figure 4B. Dans cette variante, une roue 84 en matériau déformable (e.g. en élastomère) est montée sur l'arbre 44 et comporte deux dents 86, 88. Une protubérance 82, par exemple hémisphérique, fait par ailleurs saillie du socle 84 en face de la roue 84. Lorsque l'utilisateur remonte la pipette 12 en actionnant la roue 50, la première dent 86 rencontre la protubérance 82. En augmentant le coupe appliqué à la roue 50, la dent 86 se plie, passe la protubérance 82, et reprend sa forme. Dans cette position, la dent 86 peut alors reposer sur la protubérance 82, la dureté de cette dent étant choisi pour qu'elle ne se plie pas sous l'action du poids de la pipette 12 et du porte-pipette 36. La pipette est ainsi bloquée en position haute, l'utilisateur pouvant donc relâcher la roue 50. La deuxième dent 88, de plus grande dimension (e.g. longueur et/ou largeur) nécessite pour son passage un couple beaucoup important, et définit ainsi une butée pour éviter que le porte-pipette 36 ne se déboite du socle 34, à moins que l'utilisateur déploie une force apte à casser cette dent. En variante, la protubérance 82 est remplacée par une butée comportant une bille montée sur ressort dans un logement du socle. La roue 84 peut ainsi être réalisée un matériau dur. L'action de la première dent 86 a alors pour effet de pousser la bille dans son logement, permettant ainsi le passage de la dent 86.. II est à présent décrit un procédé d'extraction de composants contenus dans un échantillon liquide à l'aide de particules magnétiques, ce procédé étant mis en œuvre à l'aide du système venant d'être décrit. Le procédé se fonde sur l'association du porte-pipette, de la pipette électronique programmable et de cônes de pipette (e.g. d'un volume de 1250 μί) afin de réaliser les différentes étapes de capture, de lavage et d'élution de particules magnétiques pour traiter un volume d'échantillon par cône de pipette compris entre 1 mL et 5 mL. La capture des particules magnétiques s'effectue séquentiellement dans les cônes de pipette au cours de cycles d'aspiration/refoulement sur l'ensemble du volume d'échantillon à traiter. A titre d'exemple, il est décrit en relation avec l'organigramme de la figure 8 un procédé de purification d'acides nucléiques viraux utilisant la chimie NucliSENS©, à savoir une extraction des acides nucléiques à l'aide de particules de silice magnétiques. A stop mechanism is also advantageously provided as illustrated in FIG. 4B. In this variant, a wheel 84 of deformable material (eg elastomer) is mounted on the shaft 44 and has two teeth 86, 88. A protrusion 82, for example hemispherical, is also projecting from the base 84 in front of the wheel 84. When the user raises the pipette 12 by actuating the wheel 50, the first tooth 86 meets the protrusion 82. By increasing the cut applied to the wheel 50, the tooth 86 bends, passes the protrusion 82, and returns to its shape . In this position, the tooth 86 can then rest on the protrusion 82, the hardness of this tooth being chosen so that it does not bend under the action of the weight of the pipette 12 and the pipette holder 36. The pipette is thus blocked in the high position, the user can therefore release the wheel 50. The second tooth 88, of larger size (eg length and / or width) requires for its passage a much larger torque, and thus defines a stop to prevent that the pipette holder 36 does not disengage the base 34, unless the user deploys a force capable of breaking this tooth. In a variant, the protuberance 82 is replaced by a stop having a ball spring-loaded in a housing of the base. The wheel 84 can thus be made of a hard material. The action of the first tooth 86 then has the effect of pushing the ball into its housing, thus allowing the passage of the tooth 86. It is now described a method for extracting components contained in a liquid sample at the same time. using magnetic particles, this method being implemented using the system just described. The process is based on the combination of the pipette holder, the programmable electronic pipette and the pipette cones (eg with a volume of 1250 μί) in order to carry out the different stages of capture, washing and elution of particles. magnets for treating a sample volume per pipette cone of between 1 mL and 5 mL. Magnetic particles are captured sequentially in the pipette tips during suction / discharge cycles over the entire volume of sample to be treated. By way of example, it is described in connection with the flowchart of FIG. 8 a process for purification of viral nucleic acids using NucliSENS © chemistry, namely a nucleic acid extraction using magnetic silica particles. .
Le procédé débute par une étape 100 de préparation des différents échantillons et réactifs nécessaires à la purification, suivie de ladite purification en 102. Notamment, la préparation 100 consiste, en 104, à mélanger l'échantillon biologique comprenant des virus dont on souhaite extraire les acides nucléiques, avec un réactif de lyse chimique des virus (e.g. le réactif de lyse « Nuclisens miniMAG » de bioMérieux, de référence 200292, ou le réactif de lyse « Nuclisens easyMAG » de bioMérieux, de référence 280130), à raison de deux volumes de réactif de lyse pour un volume d'échantillon. Le mélange est ensuite chauffé pendant 30 minutes à 56°C, libérant ainsi les acides nucléiques des virus d'une manière connue en soi. Des particules de silice magnétiques (e.g. des particules ayant un cœur paramagnétique, ferromagnétique ou ferrimagnétique présentant ou non une rémanence, recouvert d'une coque en silice), ayant pour propriété de se lier avec des acides nucléiques, sont alors introduites, en 106, dans l'échantillon lysé. The process starts with a step 100 of preparation of the different samples and reagents necessary for the purification, followed by said purification at 102. Notably, the preparation 100 consists, in 104, of mixing the biological sample comprising viruses from which it is desired to extract the nucleic acids, with a reagent for chemical lysis of viruses (eg bioMérieux's "Nuclisens miniMAG" lysis reagent, reference 200292, or bioMérieux's "Nuclisens easyMAG" lysis reagent, reference 280130), in two volumes of lysis reagent for a sample volume. The mixture is then heated for 30 minutes at 56 ° C, thereby releasing the nucleic acids from the viruses in a manner known per se. Magnetic silica particles (eg particles having a paramagnetic core, ferromagnetic or ferrimagnetic having or not a remanence, covered with a silica shell), having the property of binding with nucleic acids, are then introduced, at 106, in the lysed sample.
La préparation 100 se poursuit, en 108, par le remplissage de la microplaque 18a, ayant des puits 62 de 5mL, et des tubes d'élution PCR 69 de 0,2 mL du portoir magnétique 18b de sorte que : Preparation 100 continues, at 108, by filling the microplate 18a, having wells 62 of 5mL, and PCR elution tubes 69 of 0.2ml of the magnetic rack 18b so that:
chaque puits de la première rangée de la microplaque 18a est rempli de l'échantillon lysé comprenant les particules de silice, ci-après « échantillon lysé ». Le volume total dans chaque puits de la première rangée est de préférence supérieur à 1,5 mL en raison de l'utilisation de la microplaque Deepwell 5 mL et des volumes manipulés par la pipette électronique ; chaque puits 66 de la deuxième rangée de la microplaque 18a est rempli de 1250 μΐ, de tampon de lavage (e.g. le « NucliSENS easyMAG Extraction Buffer n°2 » de bioMérieux de référence bMx 280131) ; each well in the first row of the microplate 18a is filled with the lysed sample comprising the silica particles, hereinafter "lysed sample". The total volume in each well of the first row is preferably greater than 1.5 mL due to the use of the Deepwell 5 mL microplate and volumes handled by the electronic pipette; each well 66 of the second row of the microplate 18a is filled with 1250 μΐ of wash buffer (eg BioMérieux's "NucliSENS easyMAG Extraction Buffer No. 2" reference bMx 280131);
chaque puits 66 de la troisième rangée de la microplaque 18a est rempli de 1250 μΐ, de tampon de lavage (e.g. le « NucliSENS easyMAG Extraction Buffer n°2 » de bioMérieux de référence bMx 280131) ;  each well 66 of the third row of the microplate 18a is filled with 1250 μΐ of wash buffer (e.g. the bioMérieux NucliSENS easyMAG Extraction Buffer # 2 reference bMx 280131);
chaque tube d'élution PCR 69 logé dans le portoir magnétique 18b est rempli d'un volume de 100 de tampon d'élution (e.g. le « NucliSENS easyMAG Extraction Buffer n°3 » de bioMérieux, de référence 280132).  each PCR elution tube 69 housed in the magnetic rack 18b is filled with a volume of 100 elution buffer (e.g. the "NucliSENS easyMAG Extraction Buffer No. 3" from bioMérieux, reference 280132).
L'utilisateur place alors : The user then places:
la pipette électronique 12, avec sa rangée de cônes 20, dans le logement 38 du porte- pipette 14, en position relevée pour permettre l'introduction de la plaque 18a ; et la plaque 18a dans le logement 56 du socle 34 avec la première rangée de puits comprenant l'échantillon lysé à l'aplomb des cônes 20.  the electronic pipette 12, with its row of cones 20, in the housing 38 of the pipette holder 14, in the raised position to allow the introduction of the plate 18a; and the plate 18a in the housing 56 of the base 34 with the first row of wells comprising the lysed sample in line with the cones 20.
L'extraction 102 débute par l'homogénéisation de l'échantillon lysé. Pour se faire, la pièce aimantée 16 n'est pas placée dans le socle 34 et n'interfère donc pas avec les cônes 20. L'utilisateur tourne une des roues 50 de manière à plonger les pointes 21 des cônes 20 dans la rangée de puits de la plaque 18a comprenant l'échantillon lysé. Puis, il sélectionne à l'aide de l'interface 24 de la pipette 12 un premier protocole de pipetage comprenant au moins une phase d'aspiration/refoulement de l'échantillon lysé dans les cônes 20, et lance le protocole sélectionné. Ces phases (e.g. au nombre de deux) comprennent chacune au moins un cycle d'aspiration/refoulement (e.g. cinq cycles) suivi d'une durée d'attente de plusieurs minutes, par exemple 5 minutes. Au sens de l'invention, un cycle d'aspiration et de refoulement consiste à remplir au moins au trois quart, par exemple complètement, les cônes puis à les vider complètement, à moins qu'il ne soit spécifié autrement par le programme. Extraction 102 begins with homogenization of the lysed sample. To do this, the magnetized part 16 is not placed in the base 34 and therefore does not interfere with the cones 20. The user rotates one of the wheels 50 so as to immerse the tips 21 of the cones 20 in the row of plate well 18a comprising the lysed sample. Then, using the interface 24 of the pipette 12, it selects a first pipetting protocol comprising at least one suction / discharge phase of the lysed sample in the cones 20, and starts the selected protocol. These phases (e.g. two in number) each comprise at least one suction / discharge cycle (e.g. five cycles) followed by a waiting time of several minutes, for example 5 minutes. For the purposes of the invention, a suction and discharge cycle consists of filling at least three quarters, for example completely, the cones and emptying them completely, unless otherwise specified by the program.
Une fois l'homogénéisation terminée, les cônes 20 sont vides et leurs pointes 21 plongent dans les puits contenant l'échantillon lysé. La purification 102 se poursuit par la capture, en 112, des particules de silice de l'échantillon lysé sur la paroi interne des cônes 20. A cet effet, l'utilisateur place la pièce aimantée 16 dans le logement 78 du socle 34, sélectionne à l'aide de l'interface 24 de la pipette 12 un second protocole de pipetage puis lance le protocole sélectionné. Le second protocole comprend une pluralité de cycles d'aspiration/attente/refoulement, e.g. une dizaine de cycles, une aspiration étant séparée d'un refoulement de quelques secondes, e.g. une dizaine de secondes. A chaque aspiration et chaque refoulement, une partie des particules contenues dans l'échantillon lysé est capturée sur la paroi des cônes de pipette grâce au champ magnétique produit par la pièce aimantée 16. Les particules magnétiques, et donc également leurs acides nucléiques liés, sont ainsi capturées sous le forme de culots de particules 100 en face de la pièce aimantée 16, et de préférence sur une zone centrale à mi-hauteur des cônes 20, comme illustré à la figure 9. Une fois la capture terminée, l'échantillon lysé ayant été refoulé complètement des cônes 20 et la pièce aimantée 16 étant toujours en place, la purification 102 se poursuit par une première étape de lavage 114. A cette fin, l'utilisateur soulève le porte-pipette 14 (resp. remonte la pipette 12) de manière à libérer la plaque 18a des cônes 20, aligne la deuxième rangée de la plaque 18a avec la rangée de cône 20 puis replace le porte-pipette (resp. fait descendre la pipette) de manière à faire tremper les pointes 21 des cônes dans les puits de la plaque 18a. L'utilisateur sélectionne ensuite, à l'aide de l'interface 24, un troisième protocole de pipetage comprenant au moins une phase d'aspiration/refoulement de l'échantillon lysé dans les cônes 20, puis lance le protocole sélectionné. Le troisième protocole est par exemple identique au premier protocole. Le passage répété du tampon de lavage sur les culots de particules permet ainsi de laver ces derniers. Cette étape de lavage est avantageusement complétée, ou mise en œuvre de manière conjointe, à une modulation du champ magnétique capturant les particules sur les cônes. Par exemple, l'utilisateur fait remonter et descendre la pipette 12, ce qui a pour effet de déplacer les culots de particules sur les cônes, ou bien la pièce aimantée 16 comprend un train d'aimants permanents et l'utilisateur fait coulisser dans un mouvement de va-et-vient la pièce aimantée 16 de son logement 78, de sorte que l'intensité et les lignes de champs magnétiques capturant les culots varient, tout en conservant les particules capturées sur les cônes. La modulation du champ magnétique a ainsi pour effet de réorganiser les culots lors du lavage, et augmenter l'efficacité de celui-ci. Un second lavage est ensuite mis en œuvre en 116 à l'aide du tampon de lavage de la troisième rangée de la plaque 18a. Par exemple, les cônes sont complètement vidés du premier tampon de lavage, puis une second lavage identique au premier lavage est réalisé. Once the homogenization is complete, the cones 20 are empty and their tips 21 dive into the wells containing the lysed sample. The purification 102 is continued by the capture, at 112, of the silica particles of the lysed sample on the inner wall of the cones 20. For this purpose, the user places the magnetized part 16 in the housing 78 of the base 34, selects using the interface 24 of the pipette 12 a second pipetting protocol then starts the selected protocol. The second protocol comprises a plurality of suction cycles / waiting / repression, eg a dozen cycles, a suction being separated from a discharge of a few seconds, eg ten seconds. At each aspiration and discharge, a part of the particles contained in the lysed sample is captured on the wall of the pipette cones by virtue of the magnetic field produced by the magnetized part 16. The magnetic particles, and thus also their bound nucleic acids, are thus captured in the form of pellets of particles 100 in front of the magnetized part 16, and preferably on a central zone halfway up the cones 20, as illustrated in FIG. FIG. 9. Once the capture is complete, the lysed sample having been completely expelled from the cones 20 and the magnetic part 16 still being in place, the purification 102 continues with a first washing step 114. For this purpose, the user raises the pipette holder 14 (resp., raises the pipette 12) so as to release the plate 18a from the cones 20, aligns the second row of the plate 18a with the cone row 20 and then places the pipette holder (resp. the pipette) so as to soak the tips 21 of the cones in the wells of the plate 18a. The user then selects, using the interface 24, a third pipetting protocol comprising at least one suction / discharge phase of the lysed sample in the cones 20, then initiates the selected protocol. The third protocol is for example identical to the first protocol. The repeated passage of the washing buffer on the pellets of particles thus makes it possible to wash the latter. This washing step is advantageously completed, or implemented in conjunction with, a modulation of the magnetic field capturing the particles on the cones. For example, the user raises and lowers the pipette 12, which has the effect of moving the pellets of particles on the cones, or the magnetized part 16 comprises a train of permanent magnets and the user slides in a reciprocating the magnetic part 16 of its housing 78, so that the intensity and the magnetic field lines capturing the pellets vary, while retaining the particles captured on the cones. The modulation of the magnetic field has the effect of rearranging the pellets during washing, and increase the efficiency thereof. A second wash is then implemented at 116 using the washing buffer of the third row of the plate 18a. For example, the cones are completely emptied of the first wash buffer, then a second wash identical to the first wash is performed.
A la suite de ce second lavage, une étape de migration 118 des culots de particules 200 dans les pointes 21 des cônes 20 est mise en œuvre. Pour ce faire, les cônes 20 restent préférentiellement remplis du second tampon de lavage pour faciliter la glisse des culots 200 et restent alignés avec la seconde rangée de la plaque 18a. L'utilisateur tourne alors une des roues 50 pour faire remonter la pipette 12. La pièce aimantée 16 étant solidaire du socle 34, les culots restent donc immobiles par rapport à cette dernière et migrent vers les pointes 21 en glissant sur les parois des cônes 20 à mesure de la remontée de la pipette. L'utilisateur stoppe la remontée de la pipette 12 une fois les culots 200 dans les pointes 21, comme illustré à la figure 10, à une distance moyenne de quelques millimètres, e.g. 8 mm, des extrémités ouvertes des cônes. Dans cette position, l'utilisateur sélectionne et lance ensuite à l'aide de l'interface 24 le refoulement du tampon de lavage contenu dans les cônes 20 dans les puits de la plaque 18a. Optionnellement, une des phases de lavage, ou une phase de lavage supplémentaire consiste à retirer la pièce aimantée de manière à libérer les particules magnétiques et réaliser un lavage tout en brassant les particules dans la solution de lavage par des cycles d'aspiration et de refoulement. Les particules sont ensuite capturées une nouvelle fois en replaçant la pièce aimantée et en procédant à des cycles d'aspiration et de refoulement tel que décrit précédemment. Following this second washing, a migration step 118 of the pellets of particles 200 in the tips 21 of the cones 20 is implemented. To do this, the cones 20 are preferably filled with the second washing buffer to facilitate the sliding of the caps 200 and remain aligned with the second row of the plate 18a. The user then rotates one of the wheels 50 to raise the pipette 12. The magnetized part 16 being secured to the base 34, the caps therefore remain immobile relative to the latter and migrate to the tips 21 by sliding on the walls of the cones 20 as the pipette rises. The user stops the rise of the pipette 12 once the caps 200 in the tips 21, as shown in Figure 10, at an average distance of a few millimeters, eg 8 mm, the open ends of the cones. In this position, the user selects and then launches with the help of the interface 24 the discharge of the washing buffer contained in the cones 20 in the wells of the plate 18a. Optionally, one of the washing phases, or an additional washing phase, consists in removing the magnetized part so as to release the magnetic particles and carry out a washing while stirring the particles in the washing solution by suction and discharge cycles. . The particles are then captured again by repositioning the magnetized part and carrying out suction and discharge cycles as previously described.
La purification 102 se termine par une étape 120 de transfert dans les tubes d'élution PCR 69 des particules magnétiques présentes dans les pointes 21 des cônes 20. A cet effet, l'utilisateur soulève le porte-pipette 14, ôte la plaques 18a, place le portoir magnétique 18b dans le logement 56 de manière à aligner les tubes PCR 69 avec la rangée de cônes 20, repose le porte-pipette 14 et retire la pièce aimantée 16 du socle 14 afin de libérer les particules magnétiques capturées des cônes. Une fois les pointes 21 plongées dans les tubes 69, l'utilisateur sélectionne, à l'aide de l'interface 24, un quatrième protocole de pipetage, puis lance le protocole sélectionné. Une première variante de ce protocole consiste en des cycles d'aspiration et de refoulement du tampon d'élution dans les pointes 21 des cônes 20, ce qui permet la remise en suspension des particules magnétiques en cassant les culots de particules. Par ailleurs, la fréquence choisie pour les cycles permet, à chaque refoulement dans les tubes 69, à une parties des particules magnétiques d'être capturées dans les tubes 69 grâce au champ magnétique de la pièce aimantée 68 logée dans le portoir 64. De plus ces cycles permettent de « rincer » les pointes 21 pour récupérer des particules adhérant aux parois des cônes. Dans une deuxième variante du protocole, des cycles d'aspiration et de refoulement sont tout d'abord mis en œuvre à une fréquence plus élevée de façon à brasser plus vigoureusement le tampon et les particules, et donc d'obtenir une homogénéisation accélérée facilitant le transfert dans les tubes d'élution 69. L'étape de transfert se termine par le refoulement complet du tampon d'élution dans les tubes 69. Sous l'effet du champ magnétique du portoir 64, les particules magnétiques sont alors définitivement séparées du tampon d'élution, comme illustré à la figure 11. L'utilisateur peut ainsi récupérer les tubes 69 pour un traitement ultérieur, en particulier l'élution des acides nucléiques par chauffage, d'une manière connue en soi. The purification 102 ends with a step 120 of transfer in the PCR elution tubes 69 of the magnetic particles present in the tips 21 of the cones 20. For this purpose, the user lifts the pipette holder 14, removes the plates 18a, The magnetic rack 18b is placed in the housing 56 so as to align the PCR tubes 69 with the row of cones 20, the pipette holder 14 is rested and the magnetized piece 16 is removed from the base 14 in order to release the magnetic particles captured from the cones. Once the tips 21 are immersed in the tubes 69, the user selects, using the interface 24, a fourth pipetting protocol, then launches the selected protocol. A first variant of this protocol consists of suction and discharge cycles of the elution buffer in the tips 21 of the cones 20, which allows the resuspension of the magnetic particles by breaking the pellets of particles. Moreover, the frequency chosen for the cycles allows, at each discharge in the tubes 69, a portion of the magnetic particles to be captured in the tubes 69 by the magnetic field of the magnetized piece 68 housed in the rack 64. these cycles make it possible to "rinse" the tips 21 to recover particles adhering to the walls of the cones. In a second variant of the protocol, suction and discharge cycles are first implemented at a higher frequency so as to stir more vigorously the buffer and the particles, and thus to obtain an accelerated homogenization facilitating the transfer to the elution tubes 69. The transfer step ends with the complete discharge of the elution buffer into the tubes 69. Under the effect of the magnetic field of the rack 64, the magnetic particles are then permanently separated from the buffer Elution, as illustrated in Figure 11. The user can thus recover the tubes 69 for further processing, in particular the elution of nucleic acids by heating, in a manner known per se.
Dans le mode de réalisation du porte-pipette décrit précédemment, la pièce aimantée 16 est logée dans le socle 34. Aussi, lorsque l'utilisateur souhaite avancer la plaque 18a, il peut remonter la pipette suffisamment haut pour procéder à cette opération. Ceci provoque, comme pour la migration des particules vers les pointes, le déplacement des culots 200 sur les parois des cônes 20, ce qui présente l'avantage de « réorganiser » les culots qui peuvent rouler sur eux-mêmes. L'efficacité du lavage s'en trouve ainsi renforcée. Par contre, cela implique que l'utilisateur prend garde à ne jamais trop remonter la pipette afin d'éviter que les culots sortent des cônes. Pour ce faire, l'utilisateur peut par exemple soulever ou basculer le porte pipette pour conserver les culots à distance des ouvertures des cônes. Cette option, qui nécessite le soulèvement répété d'un dispositif dont le poids peut être important, peut cependant entraîner à long terme des troubles musculo-squelettiques. En outre, l'utilisateur doit encore prendre garde de ne pas trop soulever le porte-pipette afin d'éviter que les culots sorte des cônes. In the embodiment of the pipette holder described above, the magnetized piece 16 is housed in the base 34. Also, when the user wishes to advance the plate 18a, it can raise the pipette high enough to carry out this operation. This causes, as for the migration of particles to the tips, the displacement of the caps 200 on the walls of the cones 20, which has the advantage of "rearrange" the caps that can roll on themselves. The effectiveness of the washing is thus reinforced. However, this implies that the user is careful never to go up the pipette too much to avoid the pellets coming out of the cones. To do this, the user can for example lift or tilt the pipette holder to keep the caps away from the openings of the cones. This option, which requires the repeated lifting of a device whose weight may be important, may however lead to long-term musculoskeletal disorders. In addition, the user must be careful not to raise the pipette holder too much to prevent the pellets from coming out of the cones.
Une second mode de réalisation du porte-pipette selon l'invention permet la manipulation des plaques 18a, 18b en remontant uniquement la pipette, et donc en évitant de soulever le porte- pipette 14, tout en garantissant que les culots de particules restent à distance des pointes 21 des cônes. Ce second mode de réalisation, ainsi que les variations induites sur le procédé venant d'être décrit, sont illustrés aux figures 12 et 13. Plus particulièrement, le second mode de réalisation diffère du premier mode de réalisation par les moyens de réception de la pièce aimantée 16 dans le porte-pipette 14. Notamment, le socle 34 comprend le logement 78 pour l'insertion et le retrait de la pièce aimantée 16 comme décrit précédemment et le logement 78 est ouvert dans sa partie supérieure 130 pour permettre également l'insertion et le retrait de la pièce 16 verticalement dans le logement 78. Le support de pipette 36 comprend en outre des moyens pour fixer la pièce aimantée 16 à l'aplomb du logement ouvert 78, en particulier un ou plusieurs plots 132 en matériau aimantable (e.g. en acier) fixés sur une paroi arrière 134 du support de pipette mobile 36 (figure 12C). De cette manière, la pièce aimantée 16 est solidaire du support mobile 36, et reste en face des cônes 20 lorsque l'utilisateur remonte et descend la pipette (en particulier lors des phases de lavage), comme illustrée aux figures 12B à 13 A. A second embodiment of the pipette holder according to the invention allows the handling of the plates 18a, 18b by raising only the pipette, and therefore avoiding lifting the pipette holder 14, while ensuring that the pellets of particles remain at a distance. tips 21 of the cones. This second embodiment, as well as the variations induced on the process just described, are illustrated in FIGS. 12 and 13. More particularly, the second embodiment differs from the first embodiment by the means for receiving the workpiece. In particular, the base 34 comprises the housing 78 for the insertion and removal of the magnetized part 16 as previously described and the housing 78 is open in its upper part 130 to also allow insertion. and the removal of the piece 16 vertically in the housing 78. The pipette holder 36 further comprises means for fixing the magnetized piece 16 in line with the open housing 78, in particular one or more pads 132 of magnetizable material (eg steel) attached to a rear wall 134 of the movable pipette holder 36 (Fig. 12C). In this way, the magnetized piece 16 is integral with the movable support 36, and remains opposite the cones 20 when the user moves up and down the pipette (in particular during the washing phases), as illustrated in FIGS. 12B to 13 A.
Pour réaliser la migration des culots 200 dans les pointes des cônes 20, l'utilisateur désolidarise la pièce aimantée 16 du support de pipette 36, en appliquant en simple pression vers le bas sur la poignée 78 de la pièce 16 et remonte la pipette 12. La pièce aimantée 16 se détache des plots 134, reste donc dans le logement 78 du socle et est donc solidaire du socle 34, induisant la migration des culots 200 dans les pointes 21 des cônes comme décrit précédemment (figures 13A et 13B). Une fois la pipette remontée, l'utilisateur remplace la plaque 18a par le portoir 18b muni des tubes d'élution PCR, retire la pièce aimantée 16 et redescente la pipette (figure 13C, tubes 69 non représentés). En variante, le support de pipette 36 peut comporter un logement analogue au logement 78 du socle dans lequel l'utilisateur glisse la pièce aimantée notamment pour les phases de lavage. Il a été décrit un procédé d'extraction particulier. La présente invention s'applique cependant à tout type de capture de particules magnétiques et à tout type de séquence de pipetage. De même, il a été décrit une pipette ayant 8 canaux d'un volume particulier. La pipette peut comprend un nombre quelconques de canaux de volume quelconque en fonction de l'application visée. To carry out the migration of the pellets 200 into the tips of the cones 20, the user disconnects the magnetized piece 16 from the pipette holder 36, by simply pressing down on the handle 78 of the piece 16 and raises the pipette 12. The magnetized part 16 is detached from the studs 134, thus remains in the housing 78 of the base and is thus integral with the base 34, inducing the migration of the caps 200 in the tips 21 of the cones as previously described (Figures 13A and 13B). Once the pipette raised, the user replaces the plate 18a by the rack 18b equipped with PCR elution tubes, removes the magnetized part 16 and down the pipette (Figure 13C, tubes 69 not shown). Alternatively, the pipette holder 36 may include a housing similar to the housing 78 of the base in which the user slides the magnetized part including the wash phases. A particular extraction process has been described. The present invention, however, applies to any type of capture of magnetic particles and to any type of pipetting sequence. Similarly, a pipette having 8 channels of a particular volume has been described. The pipette may comprise any number of channels of any volume depending on the intended application.
Afin d'augmenter le nombre d'échantillons traités, deux systèmes d'extraction selon l'invention peuvent être couplés, comme illustré à la figure 14. Par exemple, les poignées de rotation 50 peuvent s'emboiter de sorte que deux extractions peuvent être menées simultanément, l'utilisateur remontant et descendant les pipettes 12 en même temps. A cette fin, les pipettes peuvent également être synchronisées, une pipette commandant par exemple l'autre pipette. In order to increase the number of processed samples, two extraction systems according to the invention can be coupled, as illustrated in FIG. 14. For example, the rotation handles 50 can be engaged so that two extractions can be conducted simultaneously, the user going up and down the pipettes 12 at the same time. To this end, the pipettes can also be synchronized, a pipette controlling for example the other pipette.
De même, il a été décrit un système d'extraction portable et semi-automatisé, particulièrement adapté aux laboratoires d'analyse ayant un nombre limité d'extractions à réaliser au quotidien. L'invention peut cependant être automatisée. Par exemple, la pipette est intégré à un automate qui comprend des mécanismes programmables de montée et de descente de la pipette et de mouvement d'aimant (ou d'activation/désactivation d'électroaimants). II a été décrit deux lavages dans les puits de la deuxième rangée et de la troisième rangée de la microplaque 18a. Le nombre de lavage peut être cependant être quelconque. De même, il a été décrit une seule étape de capture des particules dans les cônes. Une ou plusieurs étapes de libération des particules, chacune suivie d'une nouvelle étape de capture, peuvent être également prévues. Similarly, it has been described a portable and semi-automated extraction system, particularly suitable for analysis laboratories having a limited number of extractions to be performed daily. The invention can however be automated. For example, the pipette is integrated with a controller that includes programmable pipette up and down and magnet movement (or electromagnet activation / deactivation) mechanisms. Two washes have been described in the wells of the second row and the third row of the microplate 18a. The number of washings can however be any. Similarly, it has been described a single step of capturing particles in the cones. One or more particle release steps, each followed by a new capture step, may also be provided.
Pour libérer les particules, le champ magnétique de capture des particules est désactivé en retirant la pièce aimantée 16 de son logement, puis des cycles d'aspiration/refoulement d'un tampon est réalisé dans les cônes de pipette de manière à détacher les culots de particules des parois des cônes et les désagréger. Une telle procédure prend plus de 10 minutes pour détacher complètement les culots des parois des cônes avec une fréquence d'aspiration/refoulement (aspiration et refoulement complet dans les cônes) de 5 cycles par minutes. En se référant à la figure 15, une libération plus rapide d'un culot 200 est obtenue en réalisant un mouvement de va-et-vient, sur le culot 200, du ménisque 300 que forme le tampon 302 dans un cône 20. Notamment, le cycle d'aspiration/refoulement est réglé de sorte que le ménisque 300 réalise une course limitée 304 de part et d'autre du culot 200 afin d'augmenter la fréquence de passage du ménisque sur le culot. De même, la fréquence des cycles d'aspiration /refoulement est accrue pour augmenter encore ladite fréquence de passage, en particulier une fréquence cycle supérieure à 2 cycles par secondes sur la zone de présence du culot de particules magnétiques. En appliquant cette procédure, les culots se détachent des cônes en moins de 1 minute. Les inventeurs ont constaté que c'est le passage du ménisque sur un culot qui aide à détacher ce dernier. En effet, des essais ont été menés en brassant rapidement le tampon dans les cônes dans faire passer les ménisques sur les culots (i.e. « simple » mouvement de liquide devant les culots) sans gain notable de temps. Une fois la phase de libération des culots réalisée, qui a également pour effet de désagréger les culots, une phase de brassage par aspiration/refoulement complet dans les cônes est mise en œuvre (e.g. 8 cycles d'aspiration/refoulement par minute) pour terminer de désagréger les culots et homogénéiser le tampon comprenant les particules. Ces cycles d'aspiration/refoulement complets dans les puits de la microplaques brassent un plus grand volume, sur une plus grande course, ce qui facilite l'homogénéisation du tampon. In order to release the particles, the particle capture magnetic field is deactivated by withdrawing the magnetized piece 16 from its housing, and suction / discharge cycles of a tampon are carried out in the pipette cones so as to detach the pellets. particles from the cone walls and disintegrate them. Such a procedure takes more than 10 minutes to completely detach the caps from the cone walls with a suction / discharge frequency (full suction and discharge in the cones) of 5 cycles per minute. Referring to Figure 15, a faster release of a pellet 200 is achieved by reciprocating, on the cap 200, the meniscus 300 that forms the pad 302 in a cone 20. Notably, the suction / discharge cycle is adjusted so that the meniscus 300 makes a limited stroke 304 on either side of the base 200 in order to increase the frequency of passage of the meniscus on the base. Likewise, the frequency of the suction / discharge cycles is increased to further increase said passage frequency, in particular a cycle frequency greater than 2 cycles per second on the presence zone. pellet magnetic particles. By applying this procedure, the pellets detach from the cones in less than 1 minute. The inventors have found that it is the passage of the meniscus on a base that helps to detach the latter. Indeed, tests were conducted by quickly stirring the buffer in the cones in passing the meniscus on the caps (ie "simple" movement of liquid in front of the caps) without significant time gain. Once the pellets release phase has been completed, which also has the effect of breaking up the pellets, a full suction / discharge stirring phase in the cones is implemented (eg 8 suction / discharge cycles per minute) to complete to disaggregate the pellets and homogenize the buffer comprising the particles. These complete suction / discharge cycles in the wells of the microplates stir a larger volume, over a larger stroke, which facilitates the homogenization of the buffer.
Il va à présent être décrit un procédé privilégié d'extraction d'acides nucléiques (e.g. ADN et ARN), en particulier d'origine virale, par exemple à l'aide de particules de silice magnétiques. Ce procédé comprend une phase de libération des particules, e.g. telle que décrite précédemment, suivie d'une phase de lavage dans un tampon et de recapture des particules. Un gain notable de temps est obtenu ainsi qu'une extraction améliorée. En particulier, ce procédé comprend, une fois l'étape de lyse des virus effectuée : It will now be described a preferred method of extraction of nucleic acids (e.g. DNA and RNA), in particular of viral origin, for example using magnetic silica particles. This method includes a particle release phase, e.g. as previously described, followed by a buffer wash and particle recapture phase. A significant gain in time is obtained as well as improved extraction. In particular, this method comprises, once the stage of lysis of the viruses carried out:
1. une première étape de capture des particules magnétiques dans les cônes de pipette, e.g. de la manière décrite précédemment ;  1. a first step of capturing the magnetic particles in the pipette tips, e.g. as previously described;
2. suivi d'une première étape de lavage, et de préférence d'au moins une seconde étape de lavage, dans des rangées différentes de la microplaque remplies de tampon de lavage (e.g. le « NucliSENS easyMAG Extraction Buffer n°l » de bioMérieux de référence 280130). Chaque lavage comprend des cycles d'aspiration /refoulement du tampon de lavage avec les particules capturées sur les cônes de pipette, et dure au moins 15 secondes, de préférence entre 25 secondes et 35 secondes, par exemple 30 secondes, et de préférence moins d'une minute ;  2. followed by a first wash step, and preferably at least a second wash step, in different rows of the microplate filled with wash buffer (eg bioMérieux's "NucliSENS easyMAG Extraction Buffer # 1") 280130). Each wash comprises suction / discharge cycles of the wash buffer with particles captured on the pipette tips, and lasts at least 15 seconds, preferably between 25 seconds and 35 seconds, for example 30 seconds, and preferably less than 30 seconds. 'one minute ;
3. au moins une troisième étape de lavage dans une troisième rangée de la microplaque 18a remplie d'un tampon de lavage (e.gle « NucliSENS easyMAG Extraction Buffer n°2 » de bioMérieux de référence bMx 280131). Lors de cette troisième étape de lavage, les particules sont libérées en retirant l'aimant, de manière à remettre les particules en suspension, le tampon avec les particules en suspension étant aspiré/refoulé dans les puits correspondant de la microplaque 18a. La troisième étape de lavage, de préférence comprenant une phase de passage de ménisques sur les culots telle que décrite précédemment dure quelques minutes, notamment 5 minutes ;  3. at least a third washing step in a third row of the microplate 18a filled with a washing buffer (e.gle "NucliSENS easyMAG Extraction Buffer No. 2" bioMérieux reference bMx 280131). In this third washing step, the particles are released by removing the magnet, so as to resuspend the particles, the buffer with the particles in suspension being sucked / pumped into the corresponding wells of the microplate 18a. The third washing step, preferably comprising a meniscus pass phase on the pellets as described above lasts a few minutes, especially 5 minutes;
4. une deuxième étape de capture des particules sur les cônes de pipette, e.g. telle que décrite précédemment ; 5. optionnellement une quatrième étape de lavage, les particules étant capturées, dans une troisième rangée de la microplaque 18a remplie d'un tampon de lavage (e.gle « NucliSENS easyMAG Extraction Buffer n°2 » de bioMérieux de référence bMx 280131) ; 4. a second step of capturing the particles on the pipette tips, eg as described above; 5. optionally a fourth washing step, the particles being captured, in a third row of the microplate 18a filled with a washing buffer (e.gle "NucliSENS easyMAG Extraction Buffer No. 2" bioMérieux reference bMx 280131);
6. une étape de migration des culots de particules dans les pointes, suivie d'une étape de transfert dans des tubes (e.g comprenant un tampon d'élution, par exemple le « NucliSENS easyMAG Extraction Buffer n°3 » de bioMérieux, de référence 280132), e.g. tel que décrit précédemment. II a été décrit des tampons de lavage de la gamme NucliSens, en particulier des tampons d'extraction n°l, n°2 et n°3. Plus généralement :  6. a step of migration of the pellets of particles in the tips, followed by a step of transfer in tubes (eg including an elution buffer, for example the "NucliSENS easyMAG Extraction Buffer No. 3" bioMérieux, reference 280132), eg as previously described. Wash buffers of the NucliSens range have been described, in particular extraction buffers No. 1, No. 2 and No. 3. More generally :
le tampon d'extraction n°l est un tampon favorisant la capture des acides nucléiques sur la silice en créant des ponts entre les groupes silanols de la silice et les groupes phosphates des acides nucléiques. Il comprend par exemple du guanidinium thiocyanate, à savoir un agent chaotropique tel que décrit dans le document de R. Boom et al. "Rapid and simple method for purification ofi nucleic acids." Journal of Clinical Microbiology. 1989; 28 (3): 495-503 ;  extraction buffer No. 1 is a buffer promoting the capture of nucleic acids on the silica by creating bridges between the silanol groups of the silica and the phosphate groups of the nucleic acids. It comprises, for example, guanidinium thiocyanate, namely a chaotropic agent as described in the document by R. Boom et al. "Rapid and simple method for purification of nucleic acids." Journal of Clinical Microbiology. 1989; 28 (3): 495-503;
les premier et second lavages permettent d'éliminer les débris résiduels de matrice ou de micro-organismes,  the first and second washings make it possible to remove residual debris from the matrix or micro-organisms,
- le troisième et quatrième lavages permettent d'éliminer les traces de GuSCN et des inhibiteurs d'une amplification de type PCR usuellement mise en place ultérieurement sur l'ADN/ARN capturé par les particules magnétiques.  the third and fourth washings make it possible to eliminate traces of GuSCN and inhibitors of a PCR type amplification which is usually subsequently put in place on the DNA / RNA captured by the magnetic particles.
le tampon d'élution compris dans les cônes PCR permet d'enlever toute trace de tampon de lavage et d'être dans les conditions optimales pour l'étape d'élution  the elution buffer included in the PCR cones makes it possible to remove all traces of washing buffer and to be in the optimal conditions for the elution step
Le tableau suivant compare les résultats obtenus avec le dispositif selon l'invention en appliquant le protocole venant d'être décrit (2 premiers lavage suivi d'un troisième lavage avec libération des particules) en comparaison des résultats obtenus avec un dispositif de l'état de la technique, à savoir le MiniMag® commercialisé par bioMérieux et considéré comme un dispositif de référence dans l'extraction d'ARN viral. Le protocole pour le MiniMag® comprend quatre étapes de lavage avec les tampons de lavage (deux avec le tampon « NucliSENS easyMAG Extraction Buffer n°l » et deux avec le tampon « NucliSENS easyMAG Extraction Buffer n°2 »). Pour déterminer l'efficacité de l'extraction, une amplification PCR en temps réel (ou « q-PCR ») du lysat extrait est mise en œuvre et le Ct (« cycle threshold », qui quantifie un seuil de détection d'acide nucléique dans un échantillon) de chaque échantillon mesuré. Les échantillons testés en duplica sont des échantillon de 25 grammes de framboise ou d'oignon vert auquel sont ajoutés une solution de Mengo virus pure (correspondant à 500 copie du génome par 25 gramme) ou diluée à 1/10e. Invention (valeur de Ct) MiniMag® (valeur de Ct) The following table compares the results obtained with the device according to the invention by applying the protocol just described (first 2 wash followed by a third wash with release of the particles) in comparison with the results obtained with a device of the state. of the technique, namely the MiniMag® marketed by bioMérieux and considered as a reference device in the viral RNA extraction. The protocol for the MiniMag® includes four washing steps with wash buffers (two with the "NucliSENS easyMAG Extraction Buffer # 1" buffer and two with the "NucliSENS easyMAG Extraction Buffer # 2" buffer). To determine the efficiency of the extraction, a real-time PCR (or "q-PCR") amplification of the extracted lysate is implemented and the Ct ("cycle threshold", which quantifies a nucleic acid detection threshold in one sample) of each measured sample. The samples tested in duplicate are 25-gram samples of raspberry or green onion supplemented with a solution of pure Mengo virus (corresponding to 500 copies of the genome per gram) or diluted to 1/10 th . Invention (Ct value) MiniMag ® (Ct value)
Echantillon framboise Oignon vert Framboise Oignon vert pur Raspberry Sample Green Onion Raspberry Pure Green Onion
25,72 26,29 25,87 25,55 24,86 25,06  25.72 26.29 25.87 25.55 24.86 25.06
Mengo  Mengo
1/10  1/10
27,87 28,61 28,06 27,95 28,01 27,81  27.87 28.61 28.06 27.95 28.01 27.81
pur  pure
26,05 26,11 25,82 25,87 24,93 24,99  26.05 26.11 25.82 25.87 24.93 24.99
Mengo  Mengo
1/10  1/10
28,23 28,44 27,59 28,13 28,06 27,76  28.23 28.44 27.59 28.13 28.06 27.76
Comme on peut le constater, l'extraction de l'ARN viral selon l'invention donne des résultats similaires avec ceux obtenus à l'aide du MiniMag®. En outre, des tests ont été menés avec des différents lots de particules de silice magnétiques de qualité diverse. Il a été constaté que l'extraction selon l'invention est étonnamment très robuste vis-à-vis de la qualité desdites particules. En particulier, des tests ont été réalisés sur les même échantillons avec un lot de particules de moindre performance, l'extraction ne comportant pas l'étape de libération/lavage/recapture telle que décrite précédemment. Dans ce cas, le taux d'extraction était plus faible. En utilisant le procédé préféré décrit précédemment avec les particules défectueuses, des résultats similaires à ceux de la table précédente ont été obtenus. As can be seen, the extraction of the viral RNA according to the invention gives similar results with those obtained using the MiniMag®. In addition, tests were conducted with different batches of magnetic silica particles of various quality. It has been found that the extraction according to the invention is surprisingly very robust vis-à-vis the quality of said particles. In particular, tests were carried out on the same samples with a batch of particles of lesser performance, the extraction not having the release / washing / recapture step as described above. In this case, the extraction rate was lower. Using the preferred method described above with the defective particles, results similar to those of the previous table were obtained.
Il a été décrit une application de l'invention à la capture d'acides nucléiques, e.g. ARN et/ou ADN provenant d'une lyse réalisée préalablement aux phases de capture/lavage/migration et transfert. L'invention s'applique également à la capture de microorganismes (e.g. bactéries, champignons, levures) à l'aide de particules magnétiques dont la surface est fonctionnalisée pour capturer les microorganismes (e.g. recouvertes de protéines de phage ou de polycations aptes à une telle capture d'une manière connue en soi). Les particules magnétiques avec leur microorganismes capturés sont transférées dans des tubes pour subir ensuite une lyse, par exemple mécanique. Le lysat obtenu peut directement faire l'objet d'un traitement, par exemple une amplification par réaction en chaîne par polymérase (e.g. une PCR quantitative de type q-PCR), ou être purifié selon le procédé d'extraction d'acides nucléiques décrit précédemment. L'invention est particulièrement adaptée à la préparation d'échantillon microbien en vue d'une PCR. En effet, l'échantillon sur lequel est réalisé la capture de particules dans les cônes de pipette peut être de très grand volume (e.g. plusieurs millilitres) alors que le volume final des tubes dans lesquelles les particules sont transférées peuvent de très faible volume (e.g. inférieur ou égal à 200 micro litres, voire inférieur ou égal à 100 micro litres). En raison du grand volume de l'échantillon, un nombre important de microorganisme est capturé. Le passage à un très faible volume final a pour effet de concentrer les microorganisme. Ainsi, les inventeurs ont noté qu'une seule phase de capture depuis un échantillon de quelques millilitres suivie d'une seule étape de lavage, est suffisant pour obtenir des résultats par q- PCR à partir d'une lyse réalisée dans un volume de 5 microlitres. An application of the invention has been described for the capture of nucleic acids, eg RNA and / or DNA from a lysis performed prior to the capture / washing / migration and transfer phases. The invention is also applicable to the capture of microorganisms (eg bacteria, fungi, yeasts) using magnetic particles whose surface is functionalized to capture microorganisms (eg coated with phage proteins or polycations capable of such capture in a manner known per se). The magnetic particles with their captured microorganisms are transferred into tubes for subsequent lysis, for example mechanical. The lysate obtained can be directly treated, for example a polymerase chain reaction amplification (eg q-PCR quantitative PCR), or be purified according to the nucleic acid extraction method described. previously. The invention is particularly suitable for microbial sample preparation for PCR. Indeed, the sample on which the capture of particles in the pipette cones is carried out can be of very large volume (eg several milliliters) whereas the final volume of the tubes in which the particles are transferred can be of very small volume (eg less than or equal to 200 micro liters, or even less than or equal to 100 micro liters). Because of the large volume of the sample, a significant number of microorganism is captured. The transition to a very small final volume has the effect of concentrating microorganisms. Thus, the inventors have noted that a single capture phase from a sample of a few milliliters followed by a single washing step, is sufficient to obtain results by q-PCR from a lysis performed in a volume of 5 ml. microliters.
Notamment, un enrichissement de matrice alimentaire (aiguillette de poulet) en bouillon nutritif a été réalisé pendant 5h à 41 ,5°C. Un post-contamination avec une souche Salmonella Derby est réalisé à un niveau de 102 à 104 CFU/mL, ce qui correspond à des concentrations pouvant être atteintes après enrichissement en présence de pathogène dans la matrice alimentaire (i.e. des concentrations pour lesquelles un lot alimentaire est déterminé comme impropre à la consommation). Sur chaque échantillon contaminé, il est mise en œuvre, en duplica, deux procédures, une selon le protocole standardisé de capture avec le système Gene- up® de bioMérieux, France, et une selon l'invention. Notably, enrichment of food matrix (chicken needle) in nutrient broth was performed for 5 hours at 41.5 ° C. A post-contamination with a Salmonella Derby strain is carried out at a level of 10 2 to 10 4 CFU / mL, which corresponds to concentrations that can be reached after enrichment in the presence of pathogen in the food matrix (ie concentrations for which a food lot is determined to be unsuitable for consumption). On each contaminated sample, two procedures are performed in duplicate, one according to the standardized capture protocol with the Gene-up® system from bioMérieux, France, and one according to the invention.
Le protocole du Gene-up consiste en une étape de « bead-beating » de l'échantillon (i.e. disruption mécanique de la paroi des bactéries), en prélevant 20 de celui-ci que l'on met dans un tube de bead-beating contenant 180 de tampon de lavage, suivi de l'agitation pendant 5 minutes sur agitateur de microplaques pour bead-beating. 5 microlitres de la solution final sont prélevés puis font l'objet d'une q-PCR. The Gene-up protocol consists of a step of "bead-beating" of the sample (ie mechanical disruption of the bacterial wall), taking 20 of it from a bead-beating tube containing 180 wash buffer, followed by stirring for 5 minutes on microplate shaker for bead-beating. 5 microliters of the final solution are taken and then subjected to q-PCR.
Le procédé selon l'invention consiste quant à lui en : The process according to the invention consists of:
1. une étape de capture spécifique en mettant en contact 2 mL d'échantillon avec une solution de protéine de phage biotinylée (2 μg/mL finale) par :  1. a specific capture step by putting 2 mL of sample in contact with a solution of biotinylated phage protein (2 μg / mL final) by:
a. agitation par aspiration/refoulement dans les cônes de la pipette pendant 10 min. b. ajout de particules magnétiques « Hyglos Streptavidin » (50 μί) et agitation par aspiration/refoulement pendant 15 minutes (les complexes bactéries-protéines de phage biotynilées viennent se fixer sur les particules magnétiques) ;  at. suction / discharge agitation in the cones of the pipette for 10 min. b. addition of "Hyglos Streptavidin" magnetic particles (50 μl) and suction / discharge agitation for 15 minutes (the biotinylated phage bacteria-protein complexes are attached to the magnetic particles);
c. mise en place de l'aimant pour la phase de collecte des particules magnétiques dans les cônes ;  vs. setting up the magnet for the phase of collecting the magnetic particles in the cones;
d. lancement du cycle de capture des particules magnétiques ;  d. launch of the capture cycle of magnetic particles;
2. une étape de lavage en dans les puits contenant la solution de lavage TST (Tris Saline Tween) avec 5 cycles d'aspiration/refoulement ;  2. a wash step in the wells containing the TST (Tris Saline Tween) wash solution with 5 suction / discharge cycles;
3. étape de collecte des particules magnétiques dans des tubes de 5 micro litres qui font l'objet d'un traitement de bead-Beating, la solution finale de 5 microlitres faisant ensuite l'objet d'une q-PCR. Les résultats obtenus selon le protocole du Gene-up® et selon l'invention sont résumés dans la table ci-dessous: 3. Magnetic particle collection step in micro-liter tubes which are subjected to a bead-beating treatment, the final solution of 5 microliters then being subjected to q-PCR. The results obtained according to the Gene-up® protocol and according to the invention are summarized in the table below:
Le gain de sensibilité estimé est de 2 log par rapport au protocole standard Gene-UP. The estimated sensitivity gain is 2 log compared to the standard Gene-UP protocol.
La présente invention répond à une problématique de polyvalence pour l'utilisation de différentes techniques de capture magnétique (purification des acides nucléiques, Immuno- concentration magnétiques,...). Le système selon l'invention, évolutif et modulable, permet : - la réalisation d'étapes de capture/lavage/élution de particules magnétiques en utilisant un système autonome constitué par l'association d'une pipette électronique programmable et d'un support permettant la réalisation des différentes étapes précitées ; The present invention addresses a problem of versatility for the use of different magnetic capture techniques (purification of nucleic acids, magnetic immuno-concentration, etc.). The system according to the invention, which is scalable and can be modulated, makes it possible: to carry out steps of capture / washing / elution of magnetic particles by using an autonomous system constituted by the combination of a programmable electronic pipette and a support allowing carrying out the various steps mentioned above;
le traitement d'un nombre d'échantillon de 1 à 8 en fonction de la configuration de la pipette électronique utilisée ;  processing a sample number of 1 to 8 depending on the configuration of the electronic pipette used;
- la parallélisation du traitement des échantillons dans le cadre défini ci-dessus avec un système semi-automatique ;  the parallelization of the sample processing in the context defined above with a semi-automatic system;
l'association de 2 systèmes si besoin d'augmenter le nombre d'échantillon à traiter ;1 les étapes d'élution peuvent être réalisées dans différents types de tubes : tubes PCR 0.2 mL pour récupération des particules de silice magnétiques lorsqu'il s'agit de capture des acides nucléiques (e.g. chimie NucliSENS©) ou bien tubes de bead-beating dans le cas de la récupération de particules magnétiques ayant servi à la récupération de pathogènes (Immuno-Concentration Magnétique). A cette fin, le système permet la réalisation d'étapes de capture/concentration des pathogènes sur les particules magnétiques et leur lyse in-situ à l'aide de billes de céramique / verre (e.g. procédé de type CapLyse©).  the combination of 2 systems if necessary to increase the number of sample to be treated 1 the elution steps can be carried out in different types of tubes: 0.2 mL PCR tubes for recovering magnetic silica particles when it is It acts of capture of the nucleic acids (eg NucliSENS © chemistry) or of bead-beating tubes in the case of the recovery of magnetic particles used for the recovery of pathogens (Magnetic Immuno-Concentration). To this end, the system allows the realization of pathogen capture / concentration steps on the magnetic particles and their in-situ lysis using ceramic / glass beads (e.g. CapLyse © type process).

Claims

REVENDICATIONS
1. Procédé d'extraction de composants contenus dans un échantillon biologique sous forme liquide, lesdits composants étant aptes à se fixer sur des particules magnétiques, le procédé comprenant :  A method of extracting components contained in a biological sample in liquid form, said components being able to bind to magnetic particles, the method comprising:
- une phase (106) de mélange de l'échantillon avec les particules magnétiques; a phase (106) for mixing the sample with the magnetic particles;
- une phase (110) d'aspiration du mélange depuis un puits dans un cône de pipette (20) tubulaire comprenant une pointe (21) destinée au pipetage de liquide; a phase (110) of sucking the mixture from a well into a tubular pipette cone (20) comprising a tip (21) for liquid pipetting;
- une phase de capture (112) des particules magnétiques sur une paroi interne du cône de pipette (20) :  a capture phase (112) of the magnetic particles on an inner wall of the pipette cone (20):
o en appliquant un premier champ magnétique (16) au cône de pipette (20), ledit champ étant apte à attirer et maintenir les particules magnétiques dans une zone prédéterminée du cône de pipette (20), dite de « capture », au- dessus de la pointe de celui-ci ;  o by applying a first magnetic field (16) to the pipette cone (20), said field being able to attract and hold the magnetic particles in a predetermined area of the so-called "capture" pipette cone (20), above from the tip of it;
o et en appliquant au moins un cycle d'aspiration et de refoulement du mélange contenu dans le cône de pipette (20) dans un puits (62);  o and applying at least one suction and discharge cycle of the mixture contained in the pipette cone (20) in a well (62);
au moins une phase (114, 116) de lavage des particules capturées sur la paroi interne du cône de pipette (20) en :  at least one phase (114, 116) for washing the particles captured on the inner wall of the pipette cone (20) by:
o refoulant le mélange contenu dans le cône de pipette (20) ; et o en appliquant depuis un puits (62) contenant une solution de lavage au moins un cycle d'aspiration et de refoulement de la solution de lavage dans le cône de pipette (20) ;  o Forcing the mixture contained in the pipette cone (20); and o applying from a well (62) containing a wash solution at least one suction and discharge cycle of the wash solution into the pipette cone (20);
une phase (118) de migration des particules magnétiques sur la paroi interne du cône de pipette (20), depuis la zone de capture jusqu'à la pointe (21) du cône de pipette (20), en réalisant un déplacement relatif du cône de pipette (20) par rapport au premier champ magnétique (16) ;  a magnetic particle migration phase (118) on the inner wall of the pipette cone (20), from the capture zone to the tip (21) of the pipette cone (20), making a relative displacement of the cone pipette (20) relative to the first magnetic field (16);
et une phase (120) de transfert desdites particules magnétiques ayant migré dans la pointe (21) du cône de pipette (20) dans un puits de récupération (69) contenant une solution.  and a phase (120) for transferring said magnetic particles having migrated into the tip (21) of the pipette cone (20) into a solution-containing recovery well (69).
2. Procédé selon la revendication 1, dans lequel le déplacement du premier champ magnétique (16) consiste à déplacer le cône de pipette (20) parallèlement à un axe longitudinal (X) dudit cône (20), et à conserver constant le premier champ magnétique (16), l'axe longitudinal (X) du cône de pipette restant à égale distance du premier champ magnétique (16) lors du déplacement du cône de pipette (20). 2. Method according to claim 1, wherein the displacement of the first magnetic field (16) consists in moving the pipette cone (20) parallel to a longitudinal axis (X) of said cone (20), and in keeping the first field constant. magnet (16), the longitudinal axis (X) of the pipette cone remaining equidistant from the first magnetic field (16) during movement of the pipette cone (20).
3. Procédé selon la revendication 1 ou 2, dans lequel la phase (120) de transfert comprend le placement de la pointe (21) du cône de pipette (20) dans le puits de récupération (69); The method of claim 1 or 2, wherein the transfer phase (120) comprises placing the tip (21) of the pipette cone (20) in the recovery well (69);
et l'application d'un second champ magnétique (68) depuis le fond du puits de récupération (69) de manière à faire migrer dans le puits de récupération (69) les particules magnétiques contenues dans la pointe (21) du cône de pipette (20).  and applying a second magnetic field (68) from the bottom of the recovery well (69) to migrate in the recovery well (69) the magnetic particles contained in the tip (21) of the pipette cone (20).
Procédé selon la revendication 3, dans lequel le second champ magnétique (68) est produit par un aimant positionné partiellement ou entièrement sous la pointe du cône de pipette. The method of claim 3, wherein the second magnetic field (68) is produced by a magnet positioned partially or wholly under the tip of the pipette cone.
Procédé selon la revendication 3 ou 4, dans lequel le premier champ magnétique appliqué au cône de pipette est désactivé lors de l'application du second champ magnétique. The method of claim 3 or 4, wherein the first magnetic field applied to the pipette cone is disabled upon application of the second magnetic field.
Procédé selon l'une quelconque des revendications 3 à 5, dans lequel la phase (120) de transfert comporte la désactivation du premier champ magnétique (16) suivie de l'application de cycles d'aspiration et de refoulement de la solution du puits de récupération (69) dans la pointe (21) du cône de pipette, ladite application comprenant : - une première phase d'application des cycles à une première fréquence ; A method according to any one of claims 3 to 5, wherein the transfer phase (120) comprises deactivating the first magnetic field (16) followed by the application of suction and discharge cycles of the well solution. recovering (69) in the tip (21) of the pipette cone, said application comprising: - a first phase of applying the cycles at a first frequency;
suivie d'une deuxième phase d'application des cycles à une deuxième fréquence, inférieure à la première fréquence.  followed by a second phase of applying the cycles to a second frequency, lower than the first frequency.
Procédé selon l'une quelconque des revendications précédentes, comprenant, préalablement à la phase (112) de capture, une phase de brassage du mélange contenu dans le cône de pipette par l'application d'au moins un cycle d'aspiration et de refoulement dudit mélange dans le cône de pipette. Process according to any one of the preceding claims, comprising, prior to the capture phase (112), a stirring phase of the mixture contained in the pipette cone by the application of at least one suction and discharge cycle. said mixture in the pipette cone.
Procédé selon quelconque des revendications précédentes, comprenant, préalablement à la phase (120) de transfert, au moins une phase de lavage des particules capturées sur la paroi interne du cône de pipette en : Process according to any one of the preceding claims, comprising, prior to the transfer phase (120), at least one washing phase of the particles captured on the inner wall of the pipette cone by:
a. en désactivant le champ magnétique ;  at. by deactivating the magnetic field;
b. libérant les particules capturées en appliquant depuis un puits contenant une solution de lavage au moins un cycle d'aspiration et de refoulement de la solution de lavage dans le cône de pipette ;  b. releasing the captured particles by applying from at least one well containing a wash solution at least one suction and discharge cycle of the wash solution into the pipette cone;
c. en appliquant une deuxième phase de capture sur une paroi interne du cône de pipette :  vs. by applying a second capture phase on an inner wall of the pipette cone:
o en appliquant le premier champ magnétique au cône de pipette o et en appliquant au moins un cycle d'aspiration et de refoulement du mélange contenu dans le cône de pipette dans le puits contenant la solution de lavage. o Applying the first magnetic field to the pipette cone o and applying at least one suction and discharge cycle of the mixture contained in the pipette cone in the well containing the washing solution.
9. Procédé selon la revendication 8, dans lequel la libération des particules capturées comprend une phase d'application des cycles de manière à réaliser un va-et-vient d'un ménisque de ladite solution sur un culot de particules capturées dans le cône de pipette, ledit va-et-vient dudit ménisque étant réalisé sur une portion du cône inférieure à la longueur totale du cône de pipette. 9. The method of claim 8, wherein the release of the captured particles comprises a cycle application phase so as to reciprocate a meniscus of said solution on a pellet of particles captured in the cone of pipette, said reciprocating said meniscus being made on a portion of the cone less than the total length of the pipette cone.
10. Procédé selon la revendication 9, dans lequel la libération des particules capturées comprend une deuxième phase d'application des cycles de manière à aspirer et refouler totalement la solution de lavage du cône. 10. The method of claim 9, wherein the release of the captured particles comprises a second cycle application phase so as to suck up and completely discharge the cone wash solution.
11. Procédé selon la revendication 10, dans lequel la fréquence d'application des cycles de la deuxième phase est inférieure à la fréquence d'application des cycles de la première phase. 11. The method of claim 10, wherein the frequency of application of the cycles of the second phase is less than the frequency of application of the cycles of the first phase.
12. Procédé selon l'une des revendications 8 à 11, dans lequel préalablement à la libération des particules capturées au moins deux phases de lavage mises en œuvre dans deux solutions de lavage distinctes. 12. Method according to one of claims 8 to 11, wherein prior to the release of the captured particles at least two washing phases implemented in two separate washing solutions.
13. Procédé selon l'une quelconque des revendications 8 à 12, dans lequel les composants contenus dans l'échantillon biologique sont des acides nucléiques. The method of any one of claims 8 to 12, wherein the components contained in the biological sample are nucleic acids.
14. Procédé selon l'une quelconque des revendications 1 à 7, dans lequel les composants contenus dans l'échantillon biologique sont microorganismes, et dans lequel le procédé comprend une unique phase de capture et une unique phase de lavage. The method according to any one of claims 1 to 7, wherein the components contained in the biological sample are microorganisms, and wherein the method comprises a single capture phase and a single wash phase.
15. Procédé selon la revendication 14, dans lequel mélange de l'échantillon avec les particules magnétiques a un volume supérieur à 1 millilitre, et de préférence supérieur ou égal à 2 millilitres, et dans lequel le volume du puits de récupération est inférieur à 200 micro litres, et de préférence inférieur ou égal à 100 micro litres. 15. The method of claim 14, wherein mixing the sample with the magnetic particles has a volume greater than 1 milliliter, and preferably greater than or equal to 2 milliliters, and wherein the volume of the recovery well is less than 200. micro liters, and preferably less than or equal to 100 micro liters.
16. Procédé selon quelconque des revendications précédentes, comprenant, préalablement à la phase (120) de transfert, au moins une phase de lavage des particules capturées sur la paroi interne du cône de pipette en : 16. Method according to any one of the preceding claims, comprising, prior to the transfer phase (120), at least one washing phase of the particles captured on the inner wall of the pipette cone by:
en aspirant de la solution de lavage dans ledit cône de pipette ; puis en modulant le premier champ magnétique appliqué aux particules magnétiques pour capturer celles-ci sur la paroi interne du cône de pipette ; aspirating wash solution into said pipette cone; then modulating the first magnetic field applied to the magnetic particles to capture them on the inner wall of the pipette cone;
- puis en refoulant le liquide de lavage du cône de pipette.  - Then pushing the washing liquid from the pipette cone.
17. Procédé selon la revendication 16, dans lequel la modulation du premier champ magnétique est réalisé : The method of claim 16, wherein the modulation of the first magnetic field is performed:
en déplaçant le cône de pipette parallèlement à un axe longitudinal dudit cône, et en conservant constant le premier champ magnétique ;  moving the pipette cone parallel to a longitudinal axis of said cone, and keeping the first magnetic field constant;
et/ou en faisant défiler des aimants espacés les uns des autres devant les particules capturées.  and / or by scrolling magnets spaced apart from each other in front of the captured particles.
18. Procédé selon l'une quelconque des revendications précédentes, dans lequel le volume du cône de pipette est au moins dix fois supérieur au volume du puits de récupération. 18. A method according to any one of the preceding claims, wherein the volume of the pipette cone is at least ten times greater than the volume of the recovery well.
19. Procédé selon l'une quelconque des revendications précédentes, dans lequel le volume du mélange est au moins trois fois supérieur au volume du cône de pipette. The method of any of the preceding claims, wherein the volume of the mixture is at least three times greater than the volume of the pipette cone.
20. Procédé selon l'une quelconque des revendications précédentes, dans lequel les composants appartiennent au groupe formé des acides nucléiques, des microorganismes, des protéines, et des peptides. The method of any one of the preceding claims, wherein the components belong to the group consisting of nucleic acids, microorganisms, proteins, and peptides.
21. Porte-pipette (14) comprenant : Pipette holder (14) comprising:
un socle (34) ;  a base (34);
un évidement (56) formé dans le socle (34) et apte à loger de manière amovible un support de puits (18a, 18b) ;  a recess (56) formed in the base (34) and adapted to releasably receive a well support (18a, 18b);
un support de pipette (36) comprenant un premier logement (38) dans lequel est apte à être insérée une pipette (12) équipée d'au moins un cône de pipette tubulaire (20) comprenant une pointe (21) destinée au pipetage de liquide, le premier logement (38) étant ouvert sur l'évidement (56) du socle, le support de pipette (36) étant mobile en translation par rapport au socle (34) selon une direction parallèle à un axe (X) des cônes de pipette (20) et mobile entre une première position dans laquelle la pointe (21) de chaque cône de pipette est logée dans un puits (62, 69) du support de puits (18a, 18b) et au moins une deuxième position dans laquelle ladite pointe (21) est en dehors dudit puits (62, 69) ;  a pipette holder (36) comprising a first housing (38) into which is inserted a pipette (12) equipped with at least one tubular pipette cone (20) comprising a tip (21) for liquid pipetting , the first housing (38) being open on the recess (56) of the base, the pipette support (36) being movable in translation relative to the base (34) in a direction parallel to an axis (X) of the cones of pipette (20) and movable between a first position in which the tip (21) of each pipette cone is housed in a well (62, 69) of the well support (18a, 18b) and at least a second position in which said tip (21) is outside said well (62, 69);
un second logement (78) apte à loger de manière amovible une pièce aimantée (16), le second logement (78) faisant face à chacun des cônes de pipette (20) dans une position au-dessus de la pointe de celui-ci lorsque le support de pipette (36) est dans la première position, et le second logement (78) faisant face à la pointe du cône de pipette (20) lorsque le support de pipette (36) est dans la deuxième position. a second housing (78) adapted to releasably receive a magnetized piece (16), the second housing (78) facing each of the pipette tips (20) in a position above the tip thereof when the pipette holder (36) is in the first position, and the second housing (78) facing the tip of the pipette cone (20) when the pipette holder (36) is in the second position.
22. Porte-pipette selon la revendication 21, dans lequel le premier logement (38) comprend une ouverture pour l'insertion et le retrait frontaux de la pipette (12) dans le premier logement (38). The pipette holder of claim 21, wherein the first housing (38) includes an opening for front insertion and withdrawal of the pipette (12) into the first housing (38).
23. Porte-pipette selon la revendication 21 ou 22, dans lequel le second logement (78) est réalisé dans le socle (36). Pipette holder according to claim 21 or 22, wherein the second housing (78) is formed in the base (36).
24. Porte-pipette selon l'une quelconque des revendications 21 à 23, comprenant un troisième logement dans le support de pipette (36) dans lequel la pièce aimantée (16) est apte à être logé de manière amovible pour faire face à chaque cône de pipette (20) à une position au-dessus de la pointe (21) dudit cône lorsque le support de pipette (36) est dans la deuxième position. Pipette holder according to any one of claims 21 to 23, comprising a third housing in the pipette holder (36) in which the magnetized piece (16) is adapted to be removably housed to face each cone. pipette (20) at a position above the tip (21) of said cone when the pipette holder (36) is in the second position.
25. Porte-pipette selon la revendication 24, dans lequel le second et troisième logements communiquent, et dans lequel le support de pipette comprend des moyens aptes à maintenir de manière amovible la pièce aimantée dans le troisième logement. The pipette holder of claim 24, wherein the second and third housings communicate, and wherein the pipette holder comprises means for releasably holding the magnetized part in the third housing.
26. Porte-pipette selon l'une quelconque des revendications 21 à 25, dans lequel : Pipette holder according to any of claims 21 to 25, wherein:
le socle (34) comprend au moins une roue dentée (42) mobile en rotation ; et le support de pipette comprend une crémaillère (40) coopérant avec la roue dentée (42) pour translater le support de pipette (36) par rapport au socle (34) lors de la rotation de la roue dentée.  the base (34) comprises at least one toothed wheel (42) rotatable; and the pipette holder comprises a rack (40) cooperating with the gear (42) to translate the pipette holder (36) relative to the base (34) upon rotation of the gear.
27. Porte-pipette selon la revendication 26, comprenant un dispositif de verrouillage (et de déverrouillage du support de pipette (82) au moins dans la première position. The pipette holder of claim 26, comprising a locking device (and unlocking the pipette holder (82) at least in the first position.
28. Porte-pipette selon l'une des revendication 26 ou 27, comprenant au moins une poignée solidaire (50) de la roue dentée pour faire tourner celle-ci et apte à se fixer de manière amovible à une poignée solidaire (50) de la roue dentée d'un autre porte-pipette. 28. pipette holder according to one of claims 26 or 27, comprising at least one integral handle (50) of the toothed wheel to rotate it and adapted to be fixed removably to a fixed handle (50) of the gear wheel of another pipette holder.
29. Système pour l'extraction de composants contenus dans un échantillon biologique sous forme liquide, lesdits composants étant aptes à se fixer sur des particules magnétiques, le système comprenant : - une pipette (12) équipée d'au moins un cône de pipette tubulaire (20) comprenant une pointe (21) destinée au pipetage de liquide et d'un circuit d'aspiration et de refoulement dans chaque cône de pipette (20) ; 29. A system for extracting components contained in a biological sample in liquid form, said components being capable of being fixed on magnetic particles, the system comprising: a pipette (12) equipped with at least one tubular pipette cone (20) comprising a tip (21) for liquid pipetting and a suction and discharge circuit in each pipette cone (20);
au moins un support de puits (18a, 18b) ;  at least one well support (18a, 18b);
un porte-pipette (14) comprenant :  a pipette holder (14) comprising:
o un socle (34) ;  a base (34);
o un évidement (56) formé dans le socle (34) et apte à loger de manière amovible chaque support de puits ;  o a recess (56) formed in the base (34) and adapted to removably accommodate each well support;
o un support de pipette (36) comprenant un premier logement (38) dans lequel la pipette (12) est insérée, le premier logement (38) étant ouvert sur l'évidement (56) du socle (34), le support de pipette (36) étant mobile en translation par rapport au socle (34) selon une direction parallèle à un axe (X) des cônes de pipette (12) et mobile entre une première position dans laquelle la pointe (21) de chaque cône de pipette (36) est logée dans un puits (62, 69) du support de puits (18a, 18b) et au moins une deuxième position dans laquelle ladite pointe (21) est en dehors dudit puits (62, 69) ;  a pipette holder (36) comprising a first housing (38) into which the pipette (12) is inserted, the first housing (38) being open on the recess (56) of the base (34), the pipette holder (36) being movable in translation relative to the base (34) in a direction parallel to an axis (X) of the pipette cones (12) and movable between a first position in which the tip (21) of each pipette cone ( 36) is housed in a well (62, 69) of the well support (18a, 18b) and at least a second position in which said tip (21) is outside said well (62, 69);
o un second logement (78) faisant face à chacun des cônes de pipette (20) dans une position au-dessus de la pointe (21) de celui-ci lorsque le support de pipette est dans la première position, et le second logement faisant face à la pointe du cône de pipette lorsque le support de pipette est dans la deuxième position ; et  a second housing (78) facing each of the pipette tips (20) in a position above the tip (21) thereof when the pipette holder is in the first position, and the second housing making facing the tip of the pipette cone when the pipette holder is in the second position; and
une pièce aimantée (16) logée de manière amovible dans le second logement (78).  a magnetized piece (16) removably housed in the second housing (78).
30. Système selon la revendication 29, dans lequel le porte-pipette est conforme à l'une quelconque des revendications 21 à 28. The system of claim 29, wherein the pipette holder is according to any one of claims 21 to 28.
31. Support de puits (18b), comprenant une pièce (64) dans laquelle sont formés des évidements (66) pour la réception des puits (69), et au moins un aimant (68) faisant face à chacun des évidements (66) formés dans ladite pièce (64). 31. Shaft support (18b), comprising a workpiece (64) in which recesses (66) for receiving the wells (69) are formed, and at least one magnet (68) facing each of the recesses (66) formed in said piece (64).
EP16801505.5A 2016-05-03 2016-11-25 Method and system for magnetic extraction of components in a liquid sample Active EP3452589B1 (en)

Applications Claiming Priority (3)

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EP16168001.2A EP3241901B1 (en) 2016-05-03 2016-05-03 Method and system for magnetic extraction of components in a liquid sample
EP16180724 2016-07-22
PCT/EP2016/078905 WO2017190816A1 (en) 2016-05-03 2016-11-25 Method and system for magnetic extraction of components in a liquid sample

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JP2019516369A (en) 2019-06-20
CA3023112A1 (en) 2017-11-09
US11572553B2 (en) 2023-02-07
US20210115433A1 (en) 2021-04-22
WO2017190816A1 (en) 2017-11-09
EP3452589B1 (en) 2021-01-06
US20190316114A1 (en) 2019-10-17
US11584926B2 (en) 2023-02-21
ES2862393T3 (en) 2021-10-07
CN109072230A (en) 2018-12-21
JP2021063811A (en) 2021-04-22
RU2721121C1 (en) 2020-05-18
JP7482005B2 (en) 2024-05-13
CN109072230B (en) 2023-07-21
JP6861222B2 (en) 2021-04-21
KR20190003728A (en) 2019-01-09

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